Construct and Sequence for Enhanced Gene Expression

ABSTRACT

The invention relates to a method for transcription and expression using a nucleic acid construct which is characterized by the presence of a promoter followed by an intronic promoter. The invention further relates to said nucleic acid construct, an expression vector and a cell comprising said construct, and its use. The invention also relates to methods for transcription and optionally expression using a nucleotide sequence. The invention further relates to said nucleotide sequence and a construct, expression vector and cell comprising said nucleotide sequence, and its use.

FIELD OF THE INVENTION

The invention relates to a method for transcription and expression usinga nucleic acid construct which is characterized by the presence of apromoter followed by an intronic promoter. The invention further relatesto said nucleic acid construct, an expression vector and a cellcomprising said construct, and its use.

The invention also relates to methods for transcription and optionallyexpression using a nucleotide sequence. The invention further relates tosaid nucleotide sequence and a construct, expression vector and cellcomprising said nucleotide sequence, and its use.

BACKGROUND OF THE INVENTION

There is still a need in the art for alternative and preferably improvedmethods for regulating the transcription of a transcript and optionallyregulating the expression of a protein or polypeptide of interest inhost cells.

SUMMARY OF THE INVENTION

The present invention relates to a method for transcription andoptionally purifying the produced transcript comprising the steps of:

-   -   a) providing a nucleic acid construct comprising a first        promoter, a second promoter, and a nucleotide sequence of        interest, wherein said first and said second promoters are        operably linked to said nucleotide sequence of interest, and        wherein said second promoter is flanked by a first intronic        sequence located upstream of said promoter and a second intronic        sequence located downstream of said promoter; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

The present invention further relates to a method for expressing andoptionally purifying a protein or polypeptide of interest comprising thestep of:

-   -   a) providing a nucleic acid construct comprising a first        promoter, a second promoter and a nucleotide sequence encoding a        protein or polypeptide of interest, wherein said first and said        second promoters are operably linked to said nucleotide sequence        encoding a protein or polypeptide of interest, and wherein said        second promoter is flanked by a first intronic sequence located        upstream of said promoter and a second intronic sequence located        downstream of said promoter; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,        purifying said protein or polypeptide of interest.

Preferably, said first intronic sequence comprises at least a donorsplice site and said second intronic sequence comprises at least anacceptor splice site. Moreover, the nucleic acid construct of step a) ofthe method of the invention comprises the following nucleotide sequencesindicated here in their relative positions in the 5′ to 3′ direction:(i) a first promoter (ii) a first intronic sequence comprising at leasta donor splice site, (iii) a second promoter, (iv) a second intronicsequence comprising at least an acceptor splice site; and (v) anucleotide sequence encoding a protein or polypeptide of interest,wherein preferably said first promoter, said first intronic sequencecomprising at least a donor splice site, said second promoter, and saidsecond intronic sequence comprising at least an acceptor splice site areall operably linked to said nucleotide sequence encoding a protein orpolypeptide of interest.

Preferably, said first promoter has at least 50% identity to nucleotides1-969 of SEQ ID NO: 1 or nucleotides 1-614 of SEQ ID NO: 2 over itswhole length. An overview of all SEQ ID NOs is given in Table 1.Preferably, a nucleotide sequence comprising both said first promoterand said first intronic sequence comprising at least a donor splice sitehas at least 50% identity to SEQ ID NO: 1 or 2 over its whole length.Preferably, said second promoter has at least 50% sequence identity withSEQ ID NO: 57 or SEQ ID NO: 58 over its whole length.

The present invention further relates to a nucleic acid constructcomprising a first promoter and a second promoter, wherein said firstand said second promoters are configured to be both operably linked toan optional nucleotide sequence of interest, and wherein said secondpromoter is flanked by a first intronic sequence located upstream ofsaid promoter and a second intronic sequence located downstream of saidpromoter. Preferably, said first intronic sequence comprises at least adonor splice site and preferably said second intronic sequence comprisesat least an acceptor splice site. Moreover, preferably a nucleic acidconstruct of the invention comprises the following nucleotide sequencesindicated here in their relative positions in the 5′ to 3′ direction:(i) a first promoter (ii) a first intronic sequence comprising at leasta donor splice site, (iii) a second promoter, (iv) a second intronicsequence comprising at least an acceptor splice site; and optionally (v)a nucleotide sequence of interest, wherein preferably said firstpromoter, said first intronic sequence comprising at least a donorsplice site, said second promoter, and said second intronic sequencecomprising at least an acceptor splice site are all configured to beoperably linked to said optional nucleotide sequence of interest.

Preferably, said first promoter has at least 50% identity to nucleotides1-969 of SEQ ID NO: 1 or nucleotides 1-614 of SEQ ID NO: 2 over itswhole length. Preferably, a nucleotide sequence comprising both saidfirst promoter and said first intronic sequence comprising at least adonor splice site has at least 50% identity to SEQ ID NO: 1 or 2 overits whole length. Preferably, said second promoter has at least 50%sequence identity with SEQ ID NO: 57 or SEQ ID NO: 58 over its wholelength.

Preferably, said nucleic acid construct is an isolated construct.Preferably, said nucleic acid construct is a recombinant nucleic acidconstruct. Preferably, said optional nucleotide sequence of interest isa nucleotide sequence encoding a protein or polypeptide of interest.Preferably, said protein or polypeptide of interest is a heterologousprotein or polypeptide.

The present invention further relates to an expression vector comprisinga nucleic acid construct or recombinant nucleic acid construct asdefined herein.

The present invention further relates to a cell comprising a nucleicacid construct or recombinant nucleic acid construct as defined herein,and/or an expression vector as defined herein.

The present invention also relates to a use of a nucleic acid constructor recombinant nucleic acid construct as defined herein, and/or anexpression vector as defined herein and/or a cell as defined herein forthe transcription of a nucleotide sequence of interest.

The present invention further relates to a use of a nucleic acidconstruct or recombinant nucleic acid construct as defined herein,and/or an expression vector as defined herein and/or a cell as definedherein for the expression of a protein or polypeptide of interest.

The present invention further relates to a method for transcription andoptionally purifying the produced transcript comprising the step of:

-   -   a) providing a nucleic acid construct comprising an expression        enhancing element, a heterologous promoter and a nucleotide        sequence of interest of the invention, wherein said expression        enhancing element and said heterologous promoter are operably        linked to said nucleotide sequence of interest; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

The present invention further relates to a method for expressing andoptionally purifying a protein or polypeptide of interest comprising thestep of:

-   -   a) providing a nucleic acid construct comprising an expression        enhancing element, a heterologous promoter and a nucleotide        sequence encoding a protein or polypeptide of interest, wherein        said expression enhancing element and said heterologous promoter        are operably linked to said nucleotide sequence encoding a        protein or polypeptide of interest; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,    -   d) purifying said protein or polypeptide of interest.

Preferably, said nucleic acid construct of said method for transcriptionand/or expression and optionally purifying a transcript and/or proteinor polypeptide of interest further comprises an additional expressionregulating element operably linked to said nucleotide sequence ofinterest and/or said nucleotide sequence encoding a protein orpolypeptide of interest. Preferably, said additional expressionregulating element comprises an intronic sequence. A preferredadditional expression regulating element comprises or is an additionalexpression enhancing element. More preferably, said additionalexpression regulating element further comprises a translation enhancingelement.

The present invention further relates to a nucleic acid molecule that isrepresented by a nucleotide sequence comprising an expression enhancingelement of the invention, i.e. a nucleotide sequence that has at least50% identity to SEQ ID NO: 1 or 2 over its whole length. An overview ofall SEQ ID NOs is given in Table 1. Preferably, said nucleic acidmolecule is an isolated nucleic acid molecule. Preferably, said nucleicacid molecule or isolated nucleic acid molecule is represented by anucleotide sequence that has at least 50% sequence identity to SEQ IDNO: 1 or 2 over its whole length. Preferably, said nucleic acid moleculeor isolated nucleic acid molecule is represented by a nucleotidesequence comprising a sequence derived from the Cricetulus griseus genefor polyubiquitin of at most 8000 nucleotides. The present inventionfurther relates to a nucleic acid construct comprising a nucleic acidmolecule of the invention. Preferably, said nucleic acid construct isrepresented by a nucleotide sequence that further comprises aheterologous promoter, wherein preferably said expression enhancingelement and said heterologous promoter are configured to be bothoperably linked to an optional nucleotide sequence of interest.Preferably, said nucleic acid construct further comprises an additionalexpression regulating element, wherein preferably said expressionenhancing element, said heterologous promoter and said additionalexpression regulating element are configured to be all operably linkedto said optional nucleotide sequence of interest. Preferably, saidadditional expression regulating element further comprises a translationenhancing element. Preferably, said additional expression regulatingelement comprises an intronic sequence. Preferably, said optionalnucleotide sequence of interest is a nucleotide sequence encoding aprotein or polypeptide of interest. Preferably, said protein orpolypeptide of interest is a heterologous protein or polypeptide.

Preferably, said nucleic acid construct is a recombinant and/or isolatednucleic acid construct.

The present invention further relates to an expression vector comprisinga nucleic acid molecule or an isolated nucleic acid molecule as definedherein, and/or a nucleic acid construct or a recombinant and/or isolatednucleic acid construct as defined herein.

The present invention further relates to a cell comprising a nucleicacid molecule or an isolated nucleic acid molecule as defined herein,and/or a nucleic acid construct or recombinant nucleic acid construct asdefined herein, and/or an expression vector as defined herein.

The present invention also relates to a use of a nucleic acid moleculeor an isolated nucleic acid molecule as defined herein, and/or a nucleicacid construct or a recombinant and/or isolated nucleic acid constructas defined herein, and/or an expression vector as defined herein and/ora cell as defined herein for the transcription of a nucleotide sequenceof interest.

The present invention further relates to a use of a nucleic acidmolecule or an isolated nucleic acid molecule as defined herein, and/ora nucleic acid construct or a recombinant and/or isolated nucleic acidconstruct as defined herein, and/or an expression vector as definedherein and/or a cell as defined herein for the expression of a proteinor polypeptide of interest.

The present invention further relates to a method for transcription andoptionally purifying a produced transcript comprising the step of:

-   -   a) providing a nucleic acid construct comprising a nucleotide        sequence that has at least 50% identity to SEQ ID NO:88 over its        whole length and which is operably linked to a nucleotide        sequence of interest and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

The present invention further relates to a method for expressing andoptionally purifying a protein or polypeptide of interest comprising thestep of:

-   -   a) providing a nucleic acid construct comprising a nucleotide        sequence that has at least 50% identity to SEQ ID NO:88 over its        whole length and which is operably linked to a nucleotide        sequence of interest and contacting a cell with said nucleic        acid construct to obtain a transformed cell, and,    -   b) allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,    -   c) purifying said protein or polypeptide of interest.

Preferably, said nucleic acid construct of said method for transcriptionand/or expression and optionally purifying a transcript and/or proteinor polypeptide of interest further comprises an additional expressionregulating element operably linked to said nucleotide sequence ofinterest and/or said nucleotide sequence encoding a protein orpolypeptide of interest. Preferably, said additional expressionregulating element comprises an intronic sequence. Preferably, saidadditional expression regulating element further comprises a translationenhancing element.

The present invention further relates to a nucleic acid molecule that isrepresented by a nucleotide sequence that has at least 50% identity toSEQ ID NO: 88 over its whole length. Preferably, said nucleic acidmolecule is an isolated nucleic acid molecule. The present inventionfurther relates to a nucleic acid construct comprising a nucleic acidmolecule of the invention. Preferably, said nucleic acid construct isrepresented by a nucleotide sequence that further comprises an optionalnucleotide sequence of interest. Preferably, said nucleic acid constructfurther comprises an additional expression regulating element, whereinpreferably said expression enhancing element is configured to beoperably linked to said optional nucleotide sequence of interest.Preferably, said additional expression regulating element furthercomprises a translation enhancing element. Preferably, said additionalexpression regulating element comprises an intronic sequence.Preferably, said optional nucleotide sequence of interest is anucleotide sequence encoding a protein or polypeptide of interest.Preferably, said protein or polypeptide of interest is a heterologousprotein or polypeptide.

Preferably, said nucleic acid construct is a recombinant and/or isolatednucleic acid construct.

The present invention further relates to an expression vector comprisinga nucleic acid molecule or an isolated nucleic acid molecule as definedherein, and/or a nucleic acid construct or a recombinant and/or isolatednucleic acid construct as defined herein.

The present invention further relates to a cell comprising a nucleicacid molecule or an isolated nucleic acid molecule as defined herein,and/or a nucleic acid construct or recombinant nucleic acid construct asdefined herein, and/or an expression vector as defined herein.

The present invention also relates to a use of a nucleic acid moleculeor an isolated nucleic acid molecule as defined herein, and/or a nucleicacid construct or a recombinant and/or isolated nucleic acid constructas defined herein, and/or an expression vector as defined herein and/ora cell as defined herein for the transcription of a nucleotide sequenceof interest.

The present invention further relates to a use of a nucleic acidmolecule or an isolated nucleic acid molecule as defined herein, and/ora nucleic acid construct or a recombinant and/or isolated nucleic acidconstruct as defined herein, and/or an expression vector as definedherein and/or a cell as defined herein for the expression of a proteinor polypeptide of interest.

DESCRIPTION OF THE INVENTION

The inventors identified an expression construct for increasing theexpression of a protein or polypeptide of interest. The expressionconstruct of the invention is characterized by two promoters operablylinked to a coding sequence of a protein or polypeptide of interest. Anexpression construct of the invention typically comprises a firstpromoter followed by a second promoter, a coding sequence of the proteinor polypeptide of interest and a polyadenylation sequence, wherein saidsecond promoter is flanked by intronic sequences. Said promoter beingflanked by intronic sequences is denominated herein as an intronicpromoter. Additional expression regulating sequences may be insertedupstream and downstream of said first and/or second promoter and/ordownstream of the polyadenylation sequence. The inventors surprisinglyfound that an expression construct of the invention comprising apromoter followed by an intronic promoter operably linked to a codingsequence of a protein or polypeptide of interest, results in asignificant increase in expression of said protein or polypeptide ofinterest as compared to an expression construct comprising only onepromoter operably linked to said coding sequence. The inventors havefound that the expression of initially poorly expressed proteins isincreased to appreciable levels when using the combination of a promoterand intronic promoter of the invention instead of a single promoter inan expression construct encoding these proteins, as exemplified in theExamples, more specifically in Example 1. The combination of a promoterand an intronic promoter of the invention in an expression construct foran initially poorly expressed protein facilitates the generation ofclonal lines and allows for the generation of clonal lines withincreased and relevant expression levels, as exemplified in theExamples, more specifically in Example 3. Furthermore, expression ofinitially highly expressed proteins is even further increased when usingthe combination of a promoter and an intronic promoter of the inventioninstead of a single promoter in an expression construct encoding theseproteins as exemplified in the Examples, more specifically in Example 5.Furthermore, an increase in total amount of mRNA and an increase inexpression as measured on protein level was found as detailed hereinbelow and exemplified in the Examples enclosed. Furthermore, thepercentage of high-producer cell lines in a stably transfected pool issignificantly higher as compared to pools with a single promoteroperably linked to the coding sequence. As the nucleotide sequence ofthe invention comprising both a promoter and an intronic promoteroperably linked to a nucleotide sequence of interest results in anincrease in transcription, the present invention is not limited to theuse of this sequence in protein and/or polypeptide expression and/orprotein and/or polypeptide production but extends to the use of thiscombination of a promoter and intronic promoter in methods where higherlevels of transcript are desired, for instance in methods for producingnoncoding RNA transcripts as further specified herein. Furthermore, afurther benefit of the invention is that, apart from an increase intranscription level and/or increase in expression level of the proteinor polypeptide of interest, the invention allows for differenttranscripts to be formed as further detailed herein.

The inventors identified an expression enhancing element for increasingthe expression of a protein or polypeptide of interest. The presentinvention relates to said expression enhancing element. Application ofthe expression enhancing element of the invention in an expressionconstruct further comprising a heterologous promoter operably linked toa sequence encoding a protein or polypeptide of interest, results in amarked increase in expression of said protein or polypeptide of interestas compared to such expression using a similar expression constructwhich only differs to the former expression construct in that theexpression enhancing element of the invention is absent. The inventorshave found that expression of initially poorly expressed proteins isincreased to appreciable levels after insertion of the element in anexpression construct encoding these proteins as exemplified in theExamples, more specifically in Example 1. Insertion of the expressionenhancing element in an expression construct for an initially poorlyexpressed protein facilitates the generation of clonal lines and allowsfor the generation of clonal lines with relevant expression levels, asexemplified in the Examples, more specifically in Example 3.Furthermore, expression of initially highly expressed proteins is evenfurther increased after insertion of the element in an expressionconstruct encoding these proteins as exemplified in the Examples, morespecifically in Example 5. Furthermore, an increase in total amount ofmRNA level and/or an increase in expression as measured on protein levelwas found as detailed herein below and exemplified in the Examplesenclosed. As the expression enhancing element of the invention mayresult in an increase in transcription, the present invention is notlimited to the use of this element in protein and/or polypeptideexpression and/or protein and/or polypeptide production but extends tothe use of this element in methods where higher levels of transcript aredesired, for instance in methods for producing noncoding RNA transcriptsas further specified herein.

The inventors further identified a nucleic acid molecule represented bya nucleotide sequence that has at least 50% identity with SEQ ID NO: 88for increasing the expression of a protein or polypeptide of interest.The use of said nucleotide sequence is attractive as demonstrated inexample 11.

First Aspect

In a first aspect, the present invention provides a nucleic acidconstruct for increasing transcription and/or expression, comprising afirst promoter and a second promoter, which are configured to be bothoperably linked to an optional nucleotide sequence of interest within anexpression construct. “Optional” being understood herein as notnecessarily being present in an expression construct. For instance, suchnucleotide sequence of interest need not be present in a commercializedexpression vector, but may be readily introduced by a person skilled inthe art before use in a method of the invention.

Preferably, within this first aspect, said nucleotide construct of theinvention comprising a first promoter and a second promoter is capableof increasing the transcription of a nucleotide sequence of interestthat is under the control of said first promoter and second promoter.Alternatively or in combination with the increased transcription, saidnucleotide construct is also preferably capable of increasing expressionof a protein or polypeptide of interest encoded by said nucleotidesequence of interest. Preferably, transcription levels are assessed inan expression system using an expression construct comprising said firstpromoter and second promoter operably linked to a nucleotide sequence ofinterest using a suitable assay such as RT-qPCR. Preferably, within thisfirst aspect, the nucleotide construct of the invention comprising thefirst promoter and second promoter of the invention allows for anincrease in transcription of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% ofsaid nucleotide sequence of interest as compared to transcription usinga construct which only differs in that the nucleotide sequence ofinterest is under the control of a single promoter, preferably whentested in a system as exemplified in the Examples which are enclosedherein. More specifically, preferably the transcription of a nucleotidesequence of interest encoding for secreted alkaline phosphatase (SeAP)is measured in a mammalian cell system, most preferably in CHO cells,using a pcDNA3.1 expression vector comprising a first promoter andsecond promoter sequence to be tested operably linked to said nucleotidesequence of interest. Transcription is preferably measured using RT-qPCRand transcription levels are compared to transcription levels of saidnucleotide sequence of interest which are measured under the sameconditions except that said first promoter and second promoter arereplaced by a single promoter, preferably a CMV promoter represented bySEQ ID NO: 57, in the expression vector used.

Preferably, within this first aspect, expression levels are establishedin an expression system using an expression construct comprising saidfirst promoter and second promoter operably linked to a nucleotidesequence encoding a protein or polypeptide of interest. Preferably, saidprotein or polypeptide of interest is a secreted protein or polypeptideand expression of said protein or polypeptide of interest is detected bya suitable assay such as an enzyme-linked immunosorbent assay (ELISA)assay, Western blotting or, dependent on the identity of the protein orpolypeptide of interest, any suitable protein identification and/orquantification assay known to the person skilled in the art. Preferably,the first promoter and second promoter of the invention allow for anincrease in expression of protein or polypeptide expression of at least5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,500%, 1000%, 1500% or 2000% as compared to expression of said protein orpolypeptide using a construct which only differs in that the encodingsequence of the protein or polypeptide of interest is under the controlof a single promoter, preferably when tested in a system as exemplifiedin the Examples which are enclosed herein. More specifically, theexpression of nucleotide sequence of interest encoding for secretedalkaline phosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising afirst promoter and second promoter sequence to be tested operably linkedto said nucleotide sequence of interest. Expression is preferablymeasured by measuring the conversion of any suitable alkalinephosphatase substrate and expression levels are compared to expressionlevels of said nucleotide sequence of interest which are measured underthe same conditions except that the expression vector only differs inthat said first promoter and second promoter are replaced by a singlepromoter, preferably a CMV promoter represented by SEQ ID NO: 57, in theexpression vector used.

Preferably, within said first aspect, said increase of protein orpolypeptide expression is copy number independent as established by anassay suitable to determine copy number dependency by a skilled personsuch as, but not limit to, a triplex Taqman assay as further detailed inExample 4 of the present invention.

Preferably, within said first aspect, said first promoter is locatedupstream or at the 5′ site of said second promoter. Preferably, saidsecond promoter as defined herein should be devoid of sequence elementsthat will act as transcription terminators. Transcription terminatorswell known by the persons skilled in the art are sequences that canresult in premature termination of transcription such as, but notlimited to, stable hairpin structures, repeat sequences such as longterminal repeats (LTRs) or Alu repeats, polyadenylation motifs andtransposable elements.

Within the context of the first aspect of the invention a promoter is apromoter capable of initiating transcription in the host cell of choice.Promoters as used herein include tissue-specific, tissue-preferred,cell-type specific, inducible and constitutive promoters as definedherein in the Definitions section. Promoters that may be comprisedwithin said first or second promoter as defined herein are promotersthat may be employed in transcription of nucleotide sequences ofinterest and/or expression of proteins or polypeptides of interest,preferably in mammalian cells, and include, but are not limited to, thehuman or murine cytomegalovirus (CMV) promoter, a simian virus (SV40)promoter, a human or mouse ubiquitin C (UBC) promoter, a human or mouseor rat elongation factor alpha (EF1-a) promoter, mouse or hamsterbeta-actin promoter, or a hamster rpS21 promoter. The Tet-Off and Tet-Onresponsive elements upstream of a minimal promoter such as a CMVpromoter is an example of an inducible mammalian promoter. Examples ofsuitable yeast and fungal promoters are Leu2 promoter, the galactose(Gal1 or Gal7) promoter, alcohol dehydrogenase I (ADH1) promoter,glucoamylase (Gla) promoter, triose phosphate isomerase (TPI) promoter,translational elongation factor EF-I alpha (TEF2) promoter,glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter, alcoholoxidase (AOX1) promoter, or glutamate dehydrogenase (gdhA) promoter. Anexample of a strong ubiquitous promoter for expression in plants iscauliflower mosaic virus (CaMV) 35S promoter.

In an embodiment within said first aspect, said first and said secondpromoters are similar promoters. Preferably, said first promoter has atleast 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identityto said second promoter.

In another embodiment within said first aspect, said first promoter andsecond promoter are distinct or different promoters. Preferably, saidfirst promoter has less than 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% sequence identity to said secondpromoter.

In a preferred embodiment within said first aspect, said first promotersequence comprises or consists of a UBC promoter or a CCT8 promoter andsaid second promoter comprises or consists of a CMV promoter, or theother way around. Preferably, said first promoter comprises or consistsof a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity tonucleotides 1-969 of SEQ ID NO: 1 or with nucleotides 1-614 of SEQ IDNO: 2. Preferably, said second promoter comprises or consists of asequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNO: 58, or preferably to SEQ ID NO: 57 Preferred within said firstaspect is a nucleotide sequence of the invention wherein said firstpromoter comprises or consists of a sequence that has at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to nucleotides 1-969 of SEQ ID NO: 1 and saidsecond promoter comprises or consists of a sequence that has at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57.

Also preferred within said first aspect is a nucleotide sequence of theinvention wherein said first promoter comprises or consists of asequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity withnucleotides 1-614 of SEQ ID NO: 2 and said second promoter comprises orconsists of a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to SEQ ID NO: 57.

Also preferred within said first aspect is a nucleotide sequence of theinvention wherein said first promoter comprises or consists of asequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNO: 58, or preferably to SEQ ID NO: 57 and wherein said second promotercomprises or consists of a sequence that has at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to nucleotides 1-969 of SEQ ID NO 1 or withnucleotides 1-614 of SEQ ID NO 2.

Preferably within said first aspect a nucleotide sequence of theinvention wherein said first promoter comprises or consists of asequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with to SEQID NO: 57 and said second promoter comprises or consists of a sequencethat has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity nucleotides 1-969 ofSEQ ID NO: 1. Also preferred is a nucleotide sequence of the inventionwherein said first promoter comprises or consists of a sequence that hasat least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 57 and saidsecond promoter comprises or consists of a sequence that has at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity with nucleotides 1-614 of SEQ ID NO:2.

It is to be understood that within said first aspect, said first and/orsecond promoter does not consist only of a promoter enhancer sequence,such as a sequence selected from the group consisting of SEQ ID NO:52-54. Preferably, said first promoter and second promoter do notconsist only of a promoter enhancer sequence, such as a sequenceselected from the group consisting of SEQ ID NO: 52-54. Preferably, anucleotide sequence of the invention does not comprise or consist of SEQID NO: 55 or 56.

In a preferred embodiment within said first aspect, said second promoteris flanked by a first intronic sequence at the 5′ site or upstream ofsaid second promoter and a second intronic sequence at the 3′ site ordownstream of said second promoter. Being “flanked” is understood hereinas being positioned in between said indicated sequences optionallyseparated by 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-200, 1-300, 1-400,1-500, 1-600, 1-700, 1-800, 1-900, 1-1000, 1-5,000 or 1-100,000nucleotides, these nucleotides being understood to encompass the 5′-UTR.An intronic sequence is understood to be at least part of the nucleotidesequence of an intron. Preferably, said first intronic sequence at the5′ site or upstream of said second promoter comprises at least a donorsplice site or splice site GT. A donor splice site is understood hereinas a splice site that, when combined with an acceptor splice site asdefined herein, results in the formation of an intron as defined in theDefinition section. Preferably, a nucleotide sequence is an intron if atleast 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%of the primary RNA loses this sequence by RNA splicing using an assaysuitable to detect intron splicing, such as but not limited toreverse-transcriptase polymerase chain reaction (RT-PCR) followed bysize or sequence analysis of the RT-PCR. Preferred donor splice sites ofthe invention are M-W-G-[cut]-G-T-R-A-G-K or M-A-R-[cut]-G-T-R-A-G-K incase the host cell is a mammalian cell, A-G-[cut]-G-T-A-W-K in case thehost cell is a plant cell, [cut]-G-T-A-W-G-T-T in case the host cell isa yeast cell and R-G-[cut]-G-T-R-A-G, in case the host cell is an insectcell. “[cut]” is to be understood herein as the specific cut site wheresplicing will take place. Intron splicing can be assessed functionallyusing an assay as detailed in the Definition section under “intron”.Most preferably, the donor splice site comprised within the firstintronic sequence of the invention is C-T-G-[cut]-G-T-G-A-G-G orA-A-A-[cut]-G-T-G-A-G-G. Preferably, said first intronic sequenceconsists of said donor splice site or splice site GT. Preferably, saidfirst intronic sequence comprises a single donor splice site as definedherein. Preferably, said first intronic sequence is free of an acceptorsplice site as defined herein below.

Preferably, within said first aspect said second intronic sequence atthe 3′ site or downstream of said promoter comprises at least anacceptor splice site which is understood herein as the splice site AGpreferably preceded by a pyrimidine rich sequence or polypyrimidinetract nucleotide sequence, optionally separated from splice site AG by1-50 nucleotides, and optionally further comprising a branch sitecomprising the sequence Y-T-N-A-Y, at the 5′ site of the polypyrimidinetract nucleotide sequence, wherein the branch site may have thenucleotide sequence C-Y-G-A-C. An acceptor splice site is understoodherein as a splice site that, when combined with a donor splice siteencompassed within a construct, results in the formation of an intron asdefined in the Definition section. Preferably, the acceptor splice siteor splice site AG has the sequence [Y-rich]-N-Y-A-G-[cut]. Preferably,the acceptor splice site or splice site AG has the sequence[Y-rich]-N-Y-A-G-[cut]-R in case the host cell is a mammalian cell,[Y-rich]-D-Y-A-G-[cut]-R or [Y-rich]-D-Y-A-G-[cut]-R-W in case the hostcell is a plant cell, [Y-rich]-A-Y-A-G-[cut] in case the host cell is ayeast cell and [Y-rich]-N-Y-A-G-[cut] in case the host cell is an insectcell. “[Y-rich]” is to be understood herein as the polypyrimidine tractwhich is preferably defined as a consecutive sequence of at least 10nucleotides comprising at least 6, 7, 8, 9 or preferably 10 pyrimidinenucleotides. Preferably, said acceptor splice site or splice site GT hasthe sequence Y-A-G-[cut]-R. Preferably, said second intronic sequencecomprises a single acceptor splice site. In an embodiment, said secondintronic sequence is free of a donor splice site as defined herein. Inan alternative embodiment, said second intronic sequence comprises botha donor splice site and an acceptor splice site as defined herein. Mostpreferably, said second intronic sequence is an intron as defined in theDefinition section. Preferably, the second promoter and the intronicsequences flanking the second promoter are configured to form anintronic promoter (referred is to FIG. 1). An intronic promoter is knownto a person skilled in the art as a promoter located within an intronicsequence. Preferably, said intronic promoter is an intron as defined inthe Definition section. Preferably, the boundaries of the intronicpromoter of the present invention are being formed by the donor splicesite of the intronic sequence at the 5′ site or upstream of the secondpromoter of the invention and the acceptor splice site of the intronicsequence at the 3′ site or downstream of the second promoter of theinvention. The intronic promoter of the invention can have a length thatis comparable or similar to naturally occurring introns, preferablycomparable or similar to naturally occurring introns in the host cell ororganism as defined herein. Preferably, said intronic promoter asdefined herein is at most 12,000 nucleotides in length. Preferably, saidfirst intronic sequence at the 5′ site or upstream of said secondpromoter is located at the 3′ site or downstream of said first promoter.Preferably, the first promoter and second promoter, the intronicsequences flanking the second promoter, and a nucleotide sequenceencoding a protein or polypeptide of interest are configured in such away that the first promoter is upstream of the second promoter, whereinthe second promoter is flanked by said intronic sequences to form anintronic promoter, and wherein said first promoter and second promoterare configured to be both upstream and operably linked to the nucleotidesequence encoding a protein or polypeptide of interest (FIG. 1). Theintronic promoter may comprise further expression enhancing elements,but preferably the intronic promoter is free of further splice sitesapart from the donor and acceptor splice sites as defined herein withinthe first and second intronic sequences as defined herein. Preferably,one or more expression enhancing sequences are comprised within saidfirst and/or said second promoter. Without being wished to be bound byany theory, transcription starting from either of the two promoters mayresult in different transcripts (pre-mRNAs) which, upon splicing resultin different mRNAs as illustrated in FIG. 1. In support of this theory,the inventors found that different transcripts are formed using aconstruct of the invention (referred is in this respect to FIG. 1,Example 8 and FIG. 10). Furthermore, the increased activity is found tobe severely diminished by 4 nucleotides mutation in the intronicpromoter which prevents correct intron splicing (referred in thisrespect is to FIG. 9 and Example 7), also supporting the theory thatboth promoters are active in the construct. Therefore, a further benefitof the invention is that, apart from an increase in transcription of thenucleotide sequence of interest and/or an increase in expression levelof the protein or polypeptide of interest, the invention allows fordifferent transcripts to be formed. “Different transcripts” areunderstood herein as transcripts that are structurally different ordistinct, i.e. having a different or distinct nucleotide sequence.Therefore a further benefit of the invention is to direct or redirectthe splicing of a nucleotide sequence of interest. Depending on thelocation of the intronic splice sites, the transcripts may have adifferent or distinct UTR sequence and/or a different or distinct codingsequence. It is also possible that only one type of transcript isformed, e.g. in case the 5′-UTR sequences of said first and secondintronic sequences are the same. Assessment whether differenttranscripts are formed can be done using any suitable method known tothe person skilled in the art, such as but not limited to Rapidamplification of cDNA ends Polymerase Chain Reaction (RACE-PCR).

Preferably, within said first aspect, said first intronic sequence atthe 5′ site or upstream of said second promoter, has at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identitywith nucleotides 970-1449 of SEQ ID NO: 1 or at least 80%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity withnucleotides 667-1228 of SEQ ID NO: 2, preferably comprising at least adonor splice site or splice site GT.

Preferably, within said first aspect, said intronic sequence downstreamor at the 3′ site of said second promoter comprises a nucleotidesequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity with nucleotides 171-277 of SEQ IDNO: 14, nucleotides 171-274 of SEQ ID NO: 19, nucleotides 133-210 of SEQID NO: 20, nucleotides 134-211 of SEQ ID NO: 21, nucleotides 134-226 ofSEQ ID NO: 22, nucleotides 134-226 of SEQ ID NO: 23, nucleotides 133-225of SEQ ID NO: 24, nucleotides 134-226 of SEQ ID NO: 25, nucleotides146-257 of SEQ ID NO: 26, or nucleotides 147-223 of SEQ ID NO: 27,preferably comprising at least an acceptor splice site AG preceded by aTC-rich nucleotide sequence, optionally separated from splice site AG by1-50 nucleotides and a branch site comprising the sequence Y-T-N-A-Y orC-Y-G-A-C, at the 5′ site of the TC-rich nucleotide sequence.

In a preferred embodiment within said first aspect, said first promoteris flanked at its 3′ site by said first intronic sequence. In anembodiment, said first promoter and said first intronic sequence are notaligned in nature but aligned in a construct of the invention byrecombination. In another embodiment, said sequence comprising both saidfirst promoter flanked at its 3′ site by said first intronic sequence isderived from a naturally occurring sequence. In a preferred embodiment,said nucleotide sequence comprising both a first promoter and a firstintronic sequence according to the present invention is a sequencederived from the UBC ubiquitin gene. Preferably, said sequence isderived from a mammalian UBC ubiquitin gene. More preferably, saidnucleotide sequence comprising both a first promoter and a firstintronic sequence according to the present invention is derived from theCricetulus griseus homologous gene of the human UBC ubiquitin gene, saidgene being indicated as the Cricetulus sp. gene for polyubiquitin, orCRUPUQ (GenBank D63782). In a preferred embodiment, said nucleotidesequence derived from CRUPUQ is comprising both a first promoter and afirst intronic sequence of the invention and is a contiguous sequence ofat least 500, 600, 700, 800, 900, 1000 or 1117 in length, preferably atleast 1449 nucleotides in length of SEQ ID NO: 1. Preferably, saidnucleotide sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 is at most 80007000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449in length. Most preferably, said sequence being 1449 nucleotides inlength. Preferably, said nucleotide sequence is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449nucleotides in length and has at least 65% identity with SEQ ID NO: 1over its whole length. Preferably, said nucleotide sequence is at most8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500or 1449 nucleotides in length and has at least 70% identity with SEQ IDNO: 1 over its whole length. Preferably, said nucleotide sequence is atmost 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600,1500 or 1449 nucleotides in length and has at least 75% identity withSEQ ID NO: 1 over its whole length. Preferably, said nucleotide sequenceis at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700,1600, 1500 or 1449 nucleotides in length and has at least 80% identitywith SEQ ID NO: 1 over its whole length. Preferably, said nucleotidesequence is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900,1800, 1700, 1600, 1500 or 1449 nucleotides in length and has at least85% identity with SEQ ID NO: 1 over its whole length. Preferably, saidnucleotide sequence is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000,1900, 1800, 1700, 1600, 1500 or 1449 nucleotides in length and has atleast 90% identity with SEQ ID NO: 1 over its whole length. Preferably,said nucleotide sequence is at most 8000, 7000, 6000, 5000, 4000, 3000,2000, 1900, 1800, 1700, 1600, 1500 or 1449 nucleotides in length and hasat least 95% identity with SEQ ID NO: 1 over its whole length. Alsopreferred is a sequence of at most 8000 nucleotides having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to SEQ ID NO: 1.

In a further preferred embodiment within said first aspect, saidnucleotide sequence comprising both a first promoter and a firstintronic sequence according to the present invention is a sequencederived from a CCT8 gene. Preferably, said sequence is derived from amammalian CCT8 gene. More preferably, said nucleotide sequencecomprising both a first promoter and a first intronic sequence accordingto the present invention is derived from the human or Homo sapiens CCT8gene. In a preferred embodiment, said nucleotide sequence derived fromsaid CCT8 gene comprising both a first promoter and a first intronicsequence of the invention is a contiguous sequence of at least 500, 600,700, 791 or 1223 in length, preferably at least 1228 nucleotides inlength of SEQ ID NO: 2. Preferably, said nucleotide sequence having atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to SEQ ID NO: 2 is at most 8000 nucleotides in length.Preferably, said nucleotide sequence is at most 8000, 7000, 6000, 5000,4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228 in length. Mostpreferably, said sequence being 1228 nucleotides in length. Preferably,said nucleotide sequence is at most 8000, 7000, 6000, 5000, 4000, 3000,2000, 1900, 1800, 1700, 1600, 1500 or 1228 nucleotides in length and hasat least 65% identity with SEQ ID NO: 2 over its whole length.Preferably, said nucleotide sequence is at most 8000, 7000, 6000, 5000,4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228 nucleotides inlength and has at least 70% identity with SEQ ID NO: 2 over its wholelength. Preferably, said nucleotide sequence is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228nucleotides in length and has at least 75% identity with SEQ ID NO: 2over its whole length. Preferably, said nucleotide sequence is at most8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500or 1228 nucleotides in length and has at least 80% identity with SEQ IDNO: 2 over its whole length. Preferably, said nucleotide sequence is atmost 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600,1500 or 1228 nucleotides in length and has at least 85% identity withSEQ ID NO: 2 over its whole length. Preferably, said nucleotide sequenceis at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700,1600, 1500 or 1228 nucleotides in length and has at least 90% identitywith SEQ ID NO: 2 over its whole length. Preferably, said nucleotidesequence is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900,1800, 1700, 1600, 1500 or 1228 nucleotides in length and has at least95% identity with SEQ ID NO: 2 over its whole length. Also preferred isa sequence of at most 8000 nucleotides having at least 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity with SEQ ID NO: 2 over its whole length.

Preferably within said first aspect, said nucleotide sequence comprisinga first promoter and a first intronic sequence as defined herein has atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NO: 1, 2 and59-61 over its whole length. Preferably, said nucleotide sequencecomprising a first promoter and a first intronic sequence as definedherein comprises or consists of any of the sequences selected from thegroup consisting of SEQ ID NO: 1, 2 and 59-61. Most preferably, saidnucleotide sequence comprising a first promoter and a first intronicsequence as defined herein comprises or consists of any of the sequencesselected from the group consisting of SEQ ID NO: 1, 2 and 59.

Preferably, the nucleotide construct of the first aspect furthercomprises one or more additional expression regulating sequences,wherein preferably said first promoter, said intronic sequences asdefined herein, and optionally said additional expression regulatingsequence are all configured to be operably linked to an optionalnucleotide sequence of interest. An “additional expression regulatingsequence” is to be understood herein as a sequence or element inaddition to the first and/or second promoter and/or the first and/orsecond intronic sequence as defined herein above, and may be anadditional expression enhancing sequence and/or a distinct expressionenhancing sequence. An additional expression regulating sequence asencompassed by the present invention can be, but is not limited to, atranscriptional and/or translational regulation of a gene, including butnot limited to, 5′-UTR, 3′-UTR, enhancer, promoter, intron,polyadenylation signal and chromatin control elements such as S/MAR(scaffold/matrix attachment region), ubiquitous chromatin openingelement, cytosine phosphodiester guanine island and STAR (stabilizingand anti-repressor element), and any derivatives thereof. Other optionalregulating sequences that may be present in the nucleic acid constructof the invention include, but are not limited to, coding nucleotidesequences of homologous and/or heterologous nucleotide sequences,including the Iron Responsive Element (IRE), Translationalcis-Regulatory Element (TLRE) or uORFs in 5′ UTRs and poly(U) stretchesin 3′ UTRs. Such one or more additional expression regulating,preferably enhancing elements may be located on any position in theconstruct, preferably directly aligning or comprised within said firstand/or second promoter.

A further preferred regulating sequence within said first aspectcomprises or consists of a translation enhancing element. Preferably, atranslation enhancing element allows for an increase in protein orpolypeptide expression of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% ascompared to expression of said protein or polypeptide using a constructwhich only differs in that it is free of said translation enhancingelement, preferably when tested in a system as exemplified in theExamples which are enclosed herein. More specifically, preferably theexpression of nucleotide sequence of interest encoding for secretedalkaline phosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising atranslation enhancing element to be tested and a CMV promoterrepresented by SEQ ID NO: 57, operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said nucleotide sequence ofinterest which are measured under the same conditions except that theexpression vector is free of said translation enhancing element to betested.

Preferably, within said first aspect said translation enhancing elementcomprises or consists of a sequence that has at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity with any of SEQ ID NO: 3-51 over its wholelength, or a translation enhancing element that comprises or consists ofa nucleotide sequence that comprises:

-   -   i) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides;    -   ii) a TC-rich nucleotide sequence comprising at least 8        consecutive C or T nucleotides, at least 3 A-rich nucleotide        sequences comprising at least 5 consecutive A nucleotides, and a        GT-rich nucleotide sequence comprising at least 10 nucleotides,        at least 80% of which are G or T nucleotides, said first        nucleotide sequence not comprising a GAA repeat nucleotide        sequence; or,    -   iii) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides, wherein said GAA repeat nucleotide sequence is        located 3′ of any one or more of said TC-rich nucleotide        sequence, A-rich nucleotide sequences, and/or GT-rich nucleotide        sequence.

The GAA repeat nucleotide sequence is defined herein as comprising atleast 3 GAA repeats. The GAA repeat nucleotide sequence may comprise animperfect GAA repeat. The GAA repeat nucleotide sequence may have atleast 50% sequence identity, or at least 60% sequence identity, or atleast 70% sequence identity, or at least 80% sequence identity, or atleast 90% sequence identity or 100% sequence identity with nucleotides14-50 of SEQ ID NO: 3. The imperfect GAA repeat may comprise thenucleotide sequence (GAA)3ATAA(GAA)8.

The TC-rich nucleotide sequence is defined herein as having at least70%, 80%, 90% or 100% sequence identity with nucleotides 54-68 of SEQ IDNO: 3.

The A-rich nucleotide sequence is defined herein as having at least 70%,80%, 90% or 100% sequence identity with any one of nucleotides 77-87,nucleotides 93-105, nucleotides 111-121, nucleotides 126-132, ornucleotides 152-169 of SEQ ID NO: 3, respectively.

The GT-rich nucleotide sequence is defined herein as having at least70%, 80%, 90% or 100% sequence identity with nucleotides 133-148 of SEQID NO: 3.

Preferably within said first aspect, said translation enhancing sequencecomprises or consists of a sequence that has at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% sequence identity to SEQ ID NO: 19. Preferably, saidtranslation enhancing sequence is located downstream or at the 3′ siteof the second promoter sequence of the invention and upstream or at the5′ site of an optional nucleotide sequence encoding a protein orpolypeptide of interest. Preferably, said translation enhancing sequenceis located downstream or at the 3′ site of the second promoter sequenceof the invention and upstream or at the 5′ site of the second intronicsequence as defined herein.

Most preferably within said first aspect, said nucleic acid construct ofthe first aspect of the invention comprising a first promoter, a firstintronic sequence, a second promoter and a second intronic sequence, hasat least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to SEQ ID NO: 73, 74, 75, or 76.

Preferably within said first aspect, the nucleic acid construct of theinvention further comprises a nucleotide sequence of interest operablylinked to and/or under the control of said first and second promotersand optionally said additional expression regulating sequence as definedherein. It is to be understood that said first promoter and secondpromoter, and optionally said additional expression regulating sequenceare all configured to be operably linked to the same, single nucleotidesequence of interest. In a preferred embodiment, said nucleotidesequence of interest is a nucleotide sequence encoding a protein orpolypeptide of interest. The protein or polypeptide of interest can be ahomologous protein or polypeptide, but in a preferred embodiment of theinvention the protein or polypeptide of interest is a heterologousprotein or polypeptide. A nucleotide sequence encoding a heterologousprotein or polypeptide may be derived in whole or in part from anysource known to the art, including a bacterial or viral genome orepisome, eukaryotic nuclear or plasmid DNA, cDNA or chemicallysynthesized DNA. The nucleotide sequence encoding a protein orpolypeptide of interest may constitute an uninterrupted coding region orit may include one or more introns bounded by appropriate splicejunctions, it can further be composed of segments derived from differentsources, naturally occurring or synthetic. The nucleotide sequenceencoding the protein or polypeptide of interest according to the methodof the invention is preferably a full-length nucleotide sequence, butcan also be a functionally active part or other part of said full-lengthnucleotide sequence. The nucleotide sequence encoding the protein orpolypeptide of interest may also comprise signal sequences directing theprotein or polypeptide of interest when expressed to a specific locationin the cell or tissue. Furthermore, the nucleotide sequence encoding theprotein or polypeptide of interest can also comprise sequences whichfacilitate protein purification and protein detection by for instanceWestern blotting and ELISA (e.g. c-myc or polyhistidine sequences).

Within the context of the invention, the protein or polypeptide ofinterest may have industrial or medicinal (pharmaceutical) applications.Examples of proteins or polypeptides with industrial applicationsinclude enzymes such as e.g. lipases (e.g. used in the detergentindustry), proteases (used inter alia in the detergent industry, inbrewing and the like), cell wall degrading enzymes (such as, cellulases,pectinases, beta.-1,3/4- and beta.-1,6-glucanases, rhamnogalacturonases,mannanases, xylanases, pullulanases, galactanases, esterases and thelike, used in fruit processing wine making and the like or in feed),phytases, phospholipases, glycosidases (such as amylases,beta-glucosidases, arabinofuranosidases, rhamnosidases, apiosidases andthe like), dairy enzymes (e.g. chymosin). Mammalian, and preferablyhuman, proteins or polypeptides and/or enzymes with therapeutic,cosmetic or diagnostic applications include, but are not limited to,insulin, serum albumin (HSA), lactoferrin, hemoglobin a and B, tissueplasminogen activator (tPA), erythropoietin (EPO), tumor necrosisfactors (TNF), BMP (Bone Morphogenic Protein), growth factors (G-CSF,GM-CSF, M-CSF, PDGF, EGF, and the like), peptide hormones (e.g.calcitonin, somatomedin, somatotropin, growth hormones, folliclestimulating hormone (FSH), interleukins (IL-x), interferons (IFN-y),phosphatases, antibodies, and antibody-like proteins such as, but notlimited to, multispecific antibodies like DART (Dual-AffinityRe-Targeting) and Tribody protein, and antibody fragments like Fc, Fab,Fab2, Fv and scFv. Also included are bacterial and viral antigens, e.g.for use as vaccines, including e.g. heat-labile toxin B-subunit, choleratoxin B-subunit, envelope surface protein Hepatitis B virus, capsidprotein Norwalk virus, glycoprotein B Human cytomegalovirus,glycoprotein S, interferon, and transmissible gastroenteritis coronavirus receptors and the like. Further included are genes coding formutants or analogues of the said proteins.

Within the context of the invention, in an alternative embodiment, saidnucleotide sequence of interest is not a coding sequence for a proteinor a polypeptide but may be a functional nucleotide sequence such as,but is not limited to, a sequence encoding a non-coding RNA, wherein anon-coding RNA is understood to be an RNA not coding for a protein orpolypeptide. Preferably, said non-coding RNA is a reference sequence orregulatory molecule that may regulate the expression of genes orregulating the activity or localization of proteins or polypeptides. Forinstance, a non-coding RNA may be an antisense RNA or miRNA molecule. Asthe first promoter and second promoter of the invention is believed towork at the level of transcription, i.e. the increase in expression bythe sequence of the invention comprising said first promoter and secondpromoter as shown herein is believed to result from an increase intranscription, the construct of the invention can also be used forproducing increased levels of transcripts, as well as producingtranscripts with different sequences. Transcription levels can bequantified by using regular transcription quantification methods knownby the person skilled in the art such as, but not limited to, Northernblotting and RT-qPCR.

Second Aspect

In a second aspect, the present invention provides an expression vectorcomprising a nucleic acid construct according to the first aspect of theinvention. The nucleic acid construct according to the invention ispreferably a vector, in particular a plasmid, cosmid or phage ornucleotide sequence, linear or circular, of a single or double strandedDNA or RNA, derived from any source, in which a number of nucleotidesequences have been joined or recombined into a unique constructionwhich is capable of introducing any one of the nucleotide sequences ofthe invention in sense or antisense orientation into a cell. The choiceof vector is dependent on the recombinant procedures followed and thehost cell used. The vector may be an autonomously replicating vector ormay replicate together with the chromosome into which it has beenintegrated. Preferably, the vector contains a selection marker. Usefulmarkers are dependent on the host cell of choice and are well known topersons skilled in the art and are selected from, but not limited to,the selection markers as defined in third aspect of the invention. Apreferred expression vector is the pcDNA3.1 expression vector. Preferredselection markers are the neomycin resistance gene, zeocin resistancegene and blasicidin resistance gene.

Third Aspect

In a third aspect, the present invention provides a cell comprising anucleic acid construct according to the first aspect of the invention,and/or an expression vector according to the second aspect of theinvention as defined herein.

Within the context of the invention, a cell may be a mammalian,including human cell, a plant, animal, insect, fungal, yeast orbacterial cell. A recombinant host cell, such as a mammalian, includinghuman, plant, animal, insect, fungal or bacterial cell, containing oneor more copies of a nucleic acid construct according to the invention isan additional subject of the invention. By host cell is meant a cellwhich contains a nucleic acid construct such as a vector and supportsthe replication and/or expression of the nucleic acid construct.Examples of suitable bacteria are Gram positive bacteria such as severalspecies of the genera Bacillus, Streptomyces and Staphylococcus or Gramnegative bacteria such as several species of the genera Escherichia andPseudomonas. Fungal cells include yeast cells. Expression in yeast canbe achieved by using yeast strains such as Pichia pastoris,Saccharomyces cerevisiae and Hansenula polymorpha. Other fungal cells ofinterest include filamentous fungi cells as Aspergillus niger,Trichoderma reesei, and the like. Furthermore, insect cells such ascells or cell lines from Drosophila melanogaster, Spodoptera frugiperda,and Trichoplusia ni, such as, but not limited to, S2, Sf9, Sf21, andHigh Five cells, can be used as host cells. Alternatively, a suitableexpression system can be a baculovirus system or expression systemsusing mammalian cells such as CHO, COS, CPK (porcine kidney), MDCK, BHK,Sp2/0, NSO, and Vero cells. A suitable human cell or human cell line isan astrocyte, adipocyte, chondrocyte, endothelial, epithelial,fibroblast, hair, keratinocyte, melanocyte, osteoblast, skeletal muscle,smooth muscle, stem, synoviocyte cell or cell line. Examples of suitablehuman cell lines also include HEK 293 (human embryonic kidney), HeLa,Per. C6, CAP (cell lines derived from primary human amniocytes), andBowes melanoma cells. In an embodiment a human cell is not an embryonicstem cell.

Therefore, another aspect of the invention relates to a host cell thatis genetically modified, preferably by a method of the invention, inthat a host cell comprises a nucleic acid construct as herein definedabove. Host cell is a cell that has been genetically modified. Thewording host cell may be replaced by modified cell or transformed cellor recombinant cell or modified host cell or transformed host cell orrecombinant host cell. For transformation procedures in plants, suitablebacteria include Agrobacterium tumefaciens and Agrobacterium rhizogenes.

A nucleic acid construct preferably is stably maintained, either as anautonomously replicating element, or, more preferably, the nucleic acidconstruct is integrated into the host cell's genome, in which case theconstruct is usually integrated at random positions in the host cell'sgenome, for instance by non-homologous recombination. Stably transformedhost cells are produced by known methods. The term stable transformationrefers to exposing cells to methods to transfer and incorporate foreignDNA into their genome. These methods include, but are not limited totransfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

Alternatively, a protein or polypeptide of interest may be expressed ina host cell, e.g., a mammalian cell, relying on transient expressionfrom vectors.

A nucleic acid construct according to the invention preferably alsocomprises a marker gene which can provide selection or screeningcapability in a treated host cell. Selectable markers are generallypreferred for host transformation events, but are not available for allhost cells. A nucleic acid construct disclosed herein can also include anucleotide sequence encoding a marker product. A marker product can beused to determine if the construct or portion thereof has been deliveredto the cell and once delivered is being expressed. Examples of markergenes include, but are not limited to the E. coli lacZ gene, whichencodes B-galactosidase, and a gene encoding the green fluorescentprotein.

Within the context of the invention, examples of suitable selectablemarkers for mammalian cells include, but are not limited todihydrofolate reductase (DHFR), glutathione synthetase (GS), thymidinekinase, neomycin, neomycin analog G418, hygromycin, blasticidin, zeocinand puromycin.

Other suitable selectable markers include, but are not limited toantibiotic, metabolic, auxotrophic or herbicide resistant genes which,when inserted in a host cell in culture, would confer on those cells theability to withstand exposure to an antibiotic. Metabolic or auxotrophicmarker genes enable transformed cells to synthesize an essentialcomponent, usually an amino acid, which allows the cells to grow onmedia that lack this component. Another type of marker gene is one thatcan be screened by histochemical or biochemical assay, even though thegene cannot be selected for. A suitable marker gene found useful in suchhost cell transformation experience is a luciferase gene. Luciferasecatalyzes the oxidation of luciferin, resulting in the production ofoxyluciferin and light. Thus, the use of a luciferase gene provides aconvenient assay for the detection of the expression of introduced DNAin host cells by histochemical analysis of the cells. In an example of atransformation process, a nucleotide sequence sought to be expressed ina host cell could be coupled in tandem with the luciferase gene. Thetandem construct could be transformed into host cells, and the resultinghost cells could be analyzed for expression of the luciferase enzyme. Anadvantage of this marker is the non-destructive procedure of applicationof the substrate and the subsequent detection.

When such selectable markers are successfully transferred into a hostcell, the transformed host cell can survive if placed under selectivepressure. There are two widely used distinct categories of selectiveregimes. The first category is based on a cell's metabolism and the useof a mutant cell line which lacks the ability to grow independent of asupplemented media. Two non-limiting examples are CHO DHFR-cells andmouse LTK-cells. These cells lack the ability to grow without theaddition of such nutrients as thymidine or hypoxanthine. Because thesecells lack certain genes necessary for a complete nucleotide synthesispathway, they cannot survive unless the missing nucleotides are providedin a supplemented media. An alternative to supplementing the media is tointroduce an intact DHFR or TK gene into cells lacking the respectivegenes, thus altering their growth requirements. Individual cells whichwere not transformed with the DHFR or TK gene will not be capable ofsurvival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuromycin. Other useful markers are dependent on the host cell of choiceand are well known to persons skilled in the art.

When a transformed host cell is obtained with a method according to theinvention (see below), a host tissue may be regenerated from saidtransformed cell in a suitable medium, which optionally may containantibiotics or biocides known in the art for the selection oftransformed cells.

Resulting transformed host tissues are preferably identified by means ofselection using a selection marker gene as present on a nucleic acidconstruct as defined herein.

Fourth Aspect

In a fourth aspect, the present invention provides a method forexpressing and optionally purifying a protein or polypeptide of interestcomprising the step of:

-   -   a) providing a nucleic acid construct according to the first        aspect of the invention comprising a nucleotide sequence        encoding a protein or polypeptide of interest; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,    -   d) purifying said protein or polypeptide of interest.

The method of the invention may be an in vitro or ex vivo method. Themethod of the invention may be applied on a cell culture, organismculture, or tissue culture. Alternatively, next to the expression inhost cells, the protein or polypeptide of interest can be produced incell-free translation systems using RNAs derived from the nucleic acidconstructs of the present invention. The method of the invention may beperformed on cultured cells.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andplyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to express the protein orpolypeptide of interest, and optionally said protein or polypeptide issubsequently recovered. For example, the transformed cell may besubjected to conditions leading to expression of the protein orpolypeptide of interest. The person skilled in the art is well aware oftechniques to be used for expressing or overexpressing the protein orpolypeptide of interest. Methods in which the transformed cell does notneed to be subjected to specific conditions leading to expression of theprotein or polypeptide of interest, but in which the protein orpolypeptide of interest is automatically (e.g., constitutively)expressed, are also included in the method of the present invention.

Within the context of the invention, purification steps depend on theexpressed protein or polypeptide and the host cell used but can compriseisolation of the protein or polypeptide. When applied to aprotein/polypeptide, the term “isolation” indicates that the protein orpolypeptide is found in a condition other than its native environment.In a preferred form, the isolated protein or polypeptide issubstantially free of other proteins, particularly other homologousproteins. It is preferred to provide the protein or polypeptide in agreater than 40% pure form, more preferably greater than 60% pure form.Even more preferably it is preferred to provide the protein orpolypeptide in a highly purified form, i.e., greater than 80% pure, morepreferably greater than 95% pure, and even more preferably greater than99% pure, as determined by SDS-PAGE. If desired, the nucleotide sequenceencoding a protein or polypeptide of interest may be ligated to aheterologous nucleotide sequence to encode a fusion protein orpolypeptide to facilitate protein purification and protein detection onfor instance Western blot and in an ELISA. Suitable heterologoussequences include, but are not limited to, the nucleotide sequencescoding for proteins such as for instance glutathione-S-transferase,maltose binding protein, metal-binding polyhistidine, green fluorescentprotein, luciferase and beta-galactosidase. The protein or polypeptidemay also be coupled to non-peptide carriers, tags or labels thatfacilitate tracing of the protein or polypeptide, both in vivo and invitro, and allow for the identification and quantification of binding ofthe protein or polypeptide to substrates. Such labels, tags or carriersare well-known in the art and include, but are not limited to, biotin,radioactive labels and fluorescent labels.

Preferably, the method of this fourth aspect of the invention allows foran increase in expression of a protein or polypeptide of interest.Preferably, expression levels are established in an expression systemusing an expression construct according to the first aspect of theinvention comprising a first and a second promoter according to thefirst aspect of the invention, operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest. Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an ELISA assay, Western blotting or, dependent onthe identity of the protein or polypeptide of interest, any suitableprotein identification and/or quantification assay known to the personskilled in the art. Preferably, the method of the invention allows foran increase in protein or polypeptide expression of at least 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%,1000%, 1500% or 2000% as compared to a method which only differs in thata construct is used in step a) that it is free of said first promoterand said second promoter and is operably linked to a single promoter,preferably when tested in a system as exemplified in the Examples whichare enclosed herein. More specifically, the expression of nucleotidesequence of interest encoding for secreted alkaline phosphatase (SeAP)is measured in a mammalian cell system, most preferably in CHO cells,using a pcDNA3.1 expression vector comprising a first promoter andsecond promoter sequence to be tested operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said nucleotide sequence ofinterest which are measured under the same conditions except that saidfirst promoter and second promoter are replaced by a single promoter,preferably a CMV promoter represented by SEQ ID NO: 57, in theexpression vector used.

Fifth Aspect

In a fifth aspect, the present invention provides a method forexpressing a protein or polypeptide of interest in an organism,comprising the steps of:

-   -   a) providing a nucleic acid construct according to the first        aspect of the invention comprising a nucleotide sequence        encoding a protein or polypeptide of interest; and,    -   b) contacting a target cell and/or target tissue of an organism,        with said nucleic acid construct to obtain a transformed target        cell and/or transformed target tissue, allowing said transformed        cell to express the protein or polypeptide of interest; and        optionally,    -   c) allowing said transformed target cell and/or target tissue to        develop into a transformed organism; and, optionally,    -   d) allowing said transformed organism to express the protein or        polypeptide of interest, for example, subjecting said        transformed organism to conditions leading to expression of the        protein or polypeptide of interest, and optionally recovering        said protein or polypeptide.

Within the context of the invention, the target cell may be an embryonaltarget cell, e.g., embryonic stem cell, for example, derived from anon-human mammalian, such as bovine, porcine, et cetera species.Preferably, said target cell is not a human embryonic stem cell. In thecase of a multicellular fungus, such target cell may be a fungal cellthat can be proliferated into said multicellular fungus. When atransformed plant tissue or plant cell (e.g., pieces of leaf, stemsegments, roots, but also protoplasts or plant cells cultivated bysuspension) is obtained with the method according to the invention,whole plants can be regenerated from said transformed tissue or cell ina suitable medium, which optionally may contain antibiotics or biocidesknown in the art for the selection of transformed cells. The method ofthe invention may be applied in nucleic acid based vaccination and/orgene therapy preferably in a mammal, most preferably in a human.Encompassed within the present invention is a method of treatmentcomprising the method of the present aspect, wherein the protein orpolypeptide of interest is a therapeutic and/or immunogenic protein orpolypeptide. The invention also relates to a construct of the firstaspect of the invention for treatment, wherein the protein orpolypeptide of interest is a therapeutic and/or immunogenic protein orpolypeptide. Furthermore, the invention relates to the use of aconstruct of the first aspect of the invention for the manufacture of amedicament, wherein the protein or polypeptide of interest is atherapeutic and/or immunogenic protein or polypeptide.

Furthermore, a part of the invention is a non-human transformedorganism. Said organism is transformed with a nucleotide sequence,recombinant nucleic acid construct, or vector according to the presentinvention, and is capable of producing the polypeptide of interest. Thisincludes a non-human transgenic organism, such as a transgenic non-humanmammalian, transgenic plant (including propagation, harvest and tissuematerial of said transgenic plant, including, but not limited to, leafs,roots, shoots and flowers), multicellular fungus, and the like.

Preferably, the method of this fifth aspect of the invention allows foran increase in expression of a protein or polypeptide of interest insaid organism or at least in one tissue or organelle or organ of saidorganism. Preferably, expression levels are established in an expressionsystem using an expression construct according to the first aspect ofthe invention comprising a first and a second promoter according to thefirst aspect of the invention, operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest. Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an ELISA assay, Western blotting or, dependent onthe identity of the protein or polypeptide of interest, any suitableprotein identification and/or quantification assay known to the personskilled in the art. Preferably, the method of the invention allows foran increase in protein or polypeptide expression of at least 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%,1000%, 1500% or 2000% in said organism or at least in one tissue ororganelle or organ of said organism as compared to a method which onlydiffers in that a construct is used in step a) that it is free of saidfirst promoter and said second promoter and is operably linked to asingle promoter, preferably when tested in a system as exemplified inthe Examples which are enclosed herein. More specifically, theexpression of nucleotide sequence of interest encoding for secretedalkaline phosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising afirst promoter and second promoter sequence to be tested operably linkedto said nucleotide sequence of interest. Expression is preferablymeasured by measuring the conversion of any suitable alkalinephosphatase substrate and expression levels are compared to expressionlevels of said nucleotide sequence of interest which are measured underthe same conditions except that said first promoter and second promoterare replaced by a single promoter, preferably a CMV promoter representedby SEQ ID NO: 57, in the expression vector used. Preferably, saidincrease of protein or polypeptide expression is copy number independentas established by an assay suitable to determine copy number dependencyby a skilled person such as, but not limit to, a triplex Taqman assay asfurther detailed in Example 4 of the present invention.

Sixth Aspect

In a sixth aspect, the present invention provides a method fortranscription and optionally purifying the produced transcriptcomprising the step of:

-   -   a) providing a nucleic acid construct according to the first        aspect of the invention comprising a nucleotide sequence of        interest; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

In a preferred embodiment of the method according to the invention anucleic acid construct as defined above is used. The method of theinvention may be an in vitro or ex vivo method. The method of theinvention may be applied on a cell culture, organism culture, or tissueculture. The method of the invention may be applied in nucleic acidbased vaccination and/or gene therapy preferably in a mammal, preferablyin a human. Encompassed within the present invention is a method fortreatment comprising or consisting of the method of the present aspect,wherein the nucleotide sequence of interest encodes for a therapeutictranscript. The invention also relates to a construct of the firstaspect of the invention for use in treatment, wherein the nucleotidesequence of interest encodes for a therapeutic transcript. Furthermore,the invention relates to the use of a construct of the first aspect ofthe invention for the manufacture of a medicament, wherein thenucleotide sequence of interest encodes for a therapeutic transcript.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to produce a transcript ofthe nucleotide sequence of interest, and optionally the producedtranscript is subsequently recovered. For example, the transformed cellmay be subjected to conditions leading to transcription the nucleotidesequence of interest. The person skilled in the art is well aware oftechniques to be used for transcription the nucleotide sequence ofinterest. Methods in which the transformed cell does not need to besubjected to specific conditions leading to transcription of thenucleotide sequence of interest, but in which the nucleotide sequence ofinterest is automatically (e.g., constitutively) transcribed, are alsoincluded in the method of the present invention.

Purification steps depend on the transcript produced. The term“isolation” indicates that the transcript is found in a condition otherthan its native environment. In a preferred form, the isolatedtranscript is substantially free of other cellular components,particularly other homologous cellular components such as homologousproteins. It is preferred to provide the transcript in a greater than40% pure form, more preferably greater than 60% pure form. Even morepreferably it is preferred to provide the transcript in a highlypurified form, i.e., greater than 80% pure, more preferably greater than95% pure, and even more preferably greater than 99% pure, as determinedby Northern blotting.

Preferably, the method of this aspect of the invention allows for anincrease in transcription of a nucleotide sequence of interest.Preferably, transcription levels are established in an expression systemusing an expression construct according to the first aspect of theinvention comprising a first and a second promoter according to thefirst aspect of the invention operably linked to a nucleotide sequenceof interest. Preferably, transcription of said nucleotide sequence ofinterest is detected by a suitable assay such as RT-qPCR. Preferably,the method of the invention allows for an increase in transcription ofat least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,200%, 300%, 500%, 1000%, 1500% or 2000% as compared to a method whichonly differs in that a construct is used in step a) that it is free ofsaid first promoter and second promoter and is operably linked to asingle promoter, preferably when tested in a system as exemplified inthe Examples which are enclosed herein. More specifically, preferablythe transcription of a nucleotide sequence of interest encoding forsecreted alkaline phosphatase (SeAP) is measured in a mammalian cellsystem, most preferably in CHO cells, using a pcDNA3.1 expression vectorcomprising a first promoter and second promoter sequence to be testedoperably linked to said nucleotide sequence of interest. Transcriptionis preferably measured using RT-qPCR and transcription levels arecompared to transcription levels of said nucleotide sequence of interestwhich are measured under the same conditions except that said firstpromoter and second promoter are replaced by a single promoter,preferably a CMV promoter represented by SEQ ID NO: 57, in theexpression vector used.

Seventh Aspect

In a seventh aspect, the present invention provides a method forsplicing or redirecting the splicing of a nucleotide sequence ofinterest, and optionally purifying the produced transcripts comprisingthe step of:

-   -   a) providing a nucleic acid construct according to the first        aspect of the invention comprising a nucleotide sequence of        interest; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce transcripts of the        nucleotide sequence of interest resulting in the production of a        transcript; and optionally,    -   d) purifying said produced transcripts.

Preferably within this aspect, said nucleic acid construct used in stepa) comprises a nucleotide sequence upstream or at the 5′ site of thesecond intronic sequence of the invention that is different or distinctfrom the nucleotide sequence upstream or at the 5′ site of the firstintronic sequence of the invention. Preferably, the nucleotide sequencebetween first promoter and 5′ of said first intronic sequence and thenucleotide sequence between second promoter and 5′ of said secondintronic sequence differs at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100% in nucleotide sequence . Preferably, a method ofthis aspect of the invention wherein such a nucleic acid construct isused results in the production of two different or distinct transcripts,which differ in nucleotide sequence at the 5′ site of the transcripts.In case the nucleotide sequence of interest is located downstream or atthe 3′ site of the second intronic sequence, the resulting transcriptswill differ in sequence upstream of said nucleotide sequence of interestas can be detected by any suitable assay known by the person skilled inthe art, such as, but not limited to 5′RACE-PCR.

In a preferred embodiment within this aspect, the nucleotide sequence ofinterest is a sequence encoding a protein or polypeptide of interest.The method of this aspect can be used to produce two proteins orpolypeptides with different or distinct N-termini using the construct ofthe invention. Preferably, a first protein or polypeptide comprising afirst N-terminus and a second protein or polypeptide comprising a secondN-terminus are produced using the method of this aspect, whereinpreferably, a first nucleotide sequence encoding said first N-terminusis located directly upstream or at the 5′ site of said first intronicsequence and a second nucleotide sequence encoding said secondN-terminus is located directly upstream or at the 5′ site of said secondintronic sequence. Preferably said first nucleotide sequence encodingsaid first N-terminus is located downstream or at the 3′ site of saidfirst promoter and upstream or at the 5′ site of said first intronicsequence. Preferably said second nucleotide sequence encoding saidsecond N-terminus is located downstream or at the 3′ site of said secondpromoter and upstream or at the 5′ site of said second intronicsequence. Preferably, said nucleic acid construct further comprises anucleotide sequence encoding a C-terminus located downstream or at the3′ site of said second intronic sequence. In this embodiment, it isrequired that said second intronic sequence is an intron as defined inthe Definition section. The difference between the N termini may belimited to a signal sequence and result in identical expressed proteinsor polypeptides, wherein the localization of the proteins orpolypeptides may differ. If performed in an expression system as earlierdefined herein, the method of this embodiment preferably results in theproduction of two proteins or polypeptides of interest, wherein saidfirst protein or polypeptide will comprise said first N-terminus linkedto said C-terminus and said second protein or polypeptide will comprisesaid second N-terminus linked to said C-terminus, as can be detected byany suitable assay known by the person skilled in the art, such as, butnot limited to, ELISA assay, Western blotting or, dependent on theidentity of the protein or polypeptide of interest, any suitable proteinidentification and/or quantification assay known to the person skilledin the art. Preferably, said assay used to detect the two different ordistinct proteins or polypeptides produced is specifically adapted todistinguish between the different proteins or polypeptides produced, forinstance using a detecting antibody specifically binding to either thefirst or the second N-terminus of proteins or polypeptides produced.

Eight Aspect

In an eighth aspect, the present invention provides a use of a nucleicacid construct according to the first aspect of the invention, and/or ause of an expression vector according to the second aspect of theinvention, and/or a use of a cell according to the third aspect of theinvention, for the expression of a protein or polypeptide of interest.

Ninth Aspect

In a ninth aspect, the present invention provides for a nucleic acidconstruct according to the first aspect of the invention, and/or anexpression vector according to the second aspect of the invention,and/or a cell according to the third aspect of the invention for use asa medicament. The invention also relates to a method of treatmentcomprising the administration of a nucleic acid construct according tothe first aspect of the invention, and/or an expression vector accordingto the second aspect of the invention, and/or a cell according to thethird aspect of the invention, wherein preferably said administration isto a mammal, more preferably to a human. Preferably, said treatment isnucleic acid based vaccination and/or gene therapy preferably in amammal, most preferably in a human. Furthermore, the invention relatesto the use of a nucleic acid construct according to the third aspect ofthe invention, and/or the use of an expression vector according to thesecond aspect of the invention, and/or the use of a cell according tothe third aspect of the invention, for the preparation of a medicament.Preferably said medicament is for nucleic acid based vaccination and/orgene therapy preferably in a mammal, most preferably in a human.

Tenth Aspect

In a tenth aspect, the present invention provides a nucleic acidmolecule that is represented by a nucleotide sequence that comprises orconsists of an expression enhancing element for increasing transcriptionof a nucleotide sequence of interest and/or expression of a protein orpolypeptide of interest. Preferably, the expression enhancing element ofthe invention is capable of increasing the transcription of a nucleotidesequence of interest and/or expression of a protein or polypeptide ofinterest. Preferably in this aspect, the expression enhancing element ofthe invention capable of increasing the transcription of a nucleotidesequence of interest and/or expression of a protein or polypeptide ofinterest is located upstream or at the 5′ site of a promoter that isoperably linked to said nucleotide sequence of interest.

Preferably within this aspect, transcription levels are established inan expression system using an expression construct comprising saidexpression enhancing element operably linked to a nucleotide sequence ofinterest using a suitable assay such a RT-qPCR. Preferably, theexpression enhancing element of the invention allows for an increase intranscription of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared totranscription levels using a construct which only differs in that it isfree of said expression enhancing element, preferably as exemplified inthe Examples which are enclosed herein. More specifically, preferablythe transcription of a nucleotide sequence of interest encoding forsecreted alkaline phosphatase (SeAP) is measured in a mammalian cellsystem, most preferably in CHO cells, using a pcDNA3.1 expression vectorcomprising an expression enhancing element to be tested and a CMVpromoter represented by SEQ ID NO: 57, operably linked to saidnucleotide sequence of interest. Transcription is preferably measuredusing RT-qPCR and transcription levels are compared to transcriptionlevels of said nucleotide sequence of interest measured under the sameconditions except that the expression vector used is free of saidexpression enhancing element to be tested.

Preferably within this aspect, expression levels are established in anexpression system using an expression construct comprising saidexpression enhancing element operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest. Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an enzyme-linked immunosorbent assay (ELISA)assay, Western blotting or, dependent on the identity of the protein orpolypeptide of interest, any suitable protein identification and/orquantification assay known to the person skilled in the art. Preferably,the expression enhancing element of the invention allows for an increasein protein or polypeptide expression of at least 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or2000% as compared to expression of said protein or polypeptide using aconstruct which only differs in that it is free of said expressionenhancing element, preferably when tested in a system as exemplified inthe Examples which are enclosed herein. More specifically, preferablythe expression of a nucleotide sequence of interest encoding forsecreted alkaline phosphatase (SeAP) is measured in a mammalian cellsystem, most preferably in CHO cells, using a pcDNA3.1 expression vectorcomprising an expression enhancing element to be tested and a CMVpromoter represented by SEQ ID NO: 57, operably linked to saidnucleotide sequence of interest. Expression is preferably measured bymeasuring the conversion of any suitable alkaline phosphatase substrateand expression levels are compared to expression levels of saidnucleotide sequence of interest which are measured under the sameconditions except that the expression vector is free of said expressionenhancing element to be tested.

Preferably within this aspect, said nucleic acid molecule is an isolatednucleic acid molecule as defined herein. In a preferred embodiment, saidexpression enhancing element is a sequence that is derived from the UBCubiquitin gene. Preferably, said expression enhancing element is derivedfrom a mammalian UBC ubiquitin gene. More preferably, said expressionenhancing element is derived from the Cricetulus griseus homologous geneof the human UBC ubiquitin gene, said gene being indicated as theCricetulus sp. gene for polyubiquitin, or CRUPUQ (GenBank D63782).

In a preferred embodiment, said expression enhancing element derivedfrom CRUPUQ comprises or consists of a sequence that has at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to nucleotides 1-969 of SEQ ID NO: 1.Preferably, said sequence having at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to nucleotides 1-969 of SEQ ID NO: 1 is a promoter as definedin the Definition section. Preferably, said promoter is capable ofinitiating transcription of a nucleotide sequence of interest and/orexpression of a polypeptide or protein or polypeptide of interestencoded by a nucleotide sequence in a host cell as defined herein below.In a further preferred embodiment, said expression enhancing elementderived from CRUPUQ comprises or consists of an intronic sequence. Anintronic sequence is understood to be at least part of the nucleotidesequence of an intron. Preferably, said intronic sequence comprises atleast a donor splice site or splice site GT. A donor splice site isunderstood herein as a splice site that, when combined with an acceptorsplice site as defined herein, results in the formation of an intron asdefined in the Definition section. Preferably, a nucleotide sequence isan intron if at least 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% of the primary RNA loses this sequence by RNA splicingusing an assay suitable to detect intron splicing, such as but notlimited to reverse-transcriptase polymerase chain reaction (RT-PCR)followed by size or sequence analysis of the RT-PCR. Preferred donorsplice sites of the invention are M-W-G-[cut]-G-T-R-A-G-K in case thehost cell is a mammalian cell, A-G-[cut]-G-T-A-W-K in case the host cellis a plant cell, [cut]-G-T-A-W-G-T-T in case the host cell is a yeastcell and R-G-[cut]-G-T-R-A-G, in case the host cell is an insect cell.“[cut]” is to be understood herein as the specific cut site wheresplicing will take place. Intron splicing can be assessed functionallyusing an assay as detailed in the Definition section under “intron”.Most preferably, the donor splice site comprised within the expressionenhancing element of the invention is C-T-G-[cut]-G-T-G-A-G-G.Preferably, said intronic sequence encompassed within said expressionenhancing element consists of said donor splice site or splice site GT.Preferably, said intronic sequence encompassed within said expressionenhancing element is free of an acceptor splice site as defined hereinbelow. Preferably, said expression enhancing element comprising anintronic sequence has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity tonucleotides 970-1449 of SEQ ID NO: 1. In a preferred embodiment, saidexpression enhancing element comprises or consists of a promoter and anintronic sequence as defined herein. Preferably, the expressionenhancing element of the invention has at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 overits whole length. Preferably, said expression enhancing element of theinvention that has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to SEQ ID NO: 1 over its whole lengthcomprises both a promoter and an intronic sequence as defined herein.Preferably, said expression enhancing element for increasing expressionis a contiguous sequence of at least 500, 600, 700, 800, 900, 1000, 1100or 1117 in length, preferably at least 1449 nucleotides in length of SEQID NO: 1. Preferably, said expression enhancing element having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to SEQ ID NO: 1 is at most 8000 nucleotides in length.Preferably, said expression enhancing element is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449 inlength. Most preferably, said sequence being 1449 nucleotides in length.Preferably, said expression enhancing element is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449nucleotides in length and has at least 65% identity to SEQ ID NO: 1 overits whole length. Preferably, said expression enhancing element is atmost 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600,1500 or 1449 nucleotides in length and has at least 70% identity to SEQID NO: 1 over its whole length. Preferably, said expression enhancingelement is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800,1700, 1600, 1500 or 1449 nucleotides in length and has at least 75%identity to SEQ ID NO: 1 over its whole length. Preferably, saidexpression enhancing element is at most 8000, 7000, 6000, 5000, 4000,3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449 nucleotides in lengthand has at least 80% identity to SEQ ID NO: 1 over its whole length.Preferably, said expression enhancing element is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1449nucleotides in length and has at least 85% identity to SEQ ID NO: 1 overits whole length. Preferably, said expression enhancing element is atmost 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600,1500 or 1449 nucleotides in length and has at least 90% identity to SEQID NO: 1 over its whole length. Preferably, said expression enhancingelement is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800,1700, 1600, 1500 or 1449 nucleotides in length and has at least 95%identity to SEQ ID NO: 1 over its whole length. Preferably, saidexpression enhancing element comprises or consists of a sequence that isrepresented by SEQ ID NO: 1.

In a further preferred embodiment within this aspect, said expressionenhancing element of the invention is a sequence derived from the CCT8gene. Preferably, said element is derived from a mammalian CCT8 gene.More preferably, said expression enhancing element is derived from thehuman or Homo sapiens CCT8 gene.

In a preferred embodiment within said aspect, said expression enhancingelement derived from the Homo sapiens CCT8 gene comprises or consists ofa sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity tonucleotides 1-614 of SEQ ID NO: 2. Preferably, said sequence that has atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to nucleotides 1-614 of SEQ IDNO: 2 is a promoter as defined in the Definition section. In a furtherpreferred embodiment, said expression enhancing element derived from theHomo sapiens CCT8 gene comprises or consists of an intronic sequence.Preferably, said intronic sequence is an intronic sequence as earlierdefined herein comprising at least a donor splice site or splice site GTas earlier defined herein. Preferably said donor splice site has thesequence M-A-R-[cut]-G-T-R-A-G-K, most preferablyA-A-A-[cut]-G-T-G-A-G-G. Preferably, said intronic sequence encompassedwithin said expression enhancing element consists of said donor splicesite or splice site GT. Preferably, said expression enhancing elementcomprising an intronic sequence has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to nucleotides 667-1228 of SEQ ID NO: 2.

In a preferred embodiment within said aspect, said expression enhancingelement comprises or consists of a promoter and an intronic sequence asdefined herein. Preferably, the expression enhancing element of theinvention has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to SEQ ID NO: 2 over its whole length.Preferably, said expression enhancing element of the invention that hasat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to SEQ ID NO: 2 over its whole length comprises both apromoter and an intronic sequence as defined herein. Preferably, saidexpression enhancing element for increasing expression is a contiguoussequence of at least 500, 600, 700, 791 or 1223 in length, preferably atleast 1228 nucleotides in length of SEQ ID NO: 2. Preferably, saidexpression enhancing element having at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 is atmost 8000 nucleotides in length. Preferably, said expression enhancingelement is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800,1700, 1600, 1500 or 1228 in length. Most preferably, said sequence being1228 nucleotides in length. Preferably, said expression enhancingelement is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800,1700, 1600, 1500 or 1228 nucleotides in length and has at least 65%identity to SEQ ID NO: 2 over its whole length. Preferably, saidexpression enhancing element is at most 8000, 7000, 6000, 5000, 4000,3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228 nucleotides in lengthand has at least 70% identity to SEQ ID NO: 2 over its whole length.Preferably, said expression enhancing element is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228nucleotides in length and has at least 75% identity to SEQ ID NO: 2 overits whole length. Preferably, said expression enhancing element is atmost 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600,1500 or 1228 nucleotides in length and has at least 80% identity to SEQID NO: 2 over its whole length. Preferably, said expression enhancingelement is at most 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1900, 1800,1700, 1600, 1500 or 1228 nucleotides in length and has at least 85%identity to SEQ ID NO: 2 over its whole length. Preferably, saidexpression enhancing element is at most 8000, 7000, 6000, 5000, 4000,3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228 nucleotides in lengthand has at least 90% identity to SEQ ID NO: 2 over its whole length.Preferably, said expression enhancing element is at most 8000, 7000,6000, 5000, 4000, 3000, 2000, 1900, 1800, 1700, 1600, 1500 or 1228nucleotides in length and has at least 95% identity to SEQ ID NO: 2 overits whole length. Preferably, said expression enhancing elementcomprises or consists of a sequence that is represented by SEQ ID NO: 2.

Further preferred is a nucleotide sequence comprising an expressionenhancing element as defined herein that has at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to any of SEQ ID NO: 59-61 over its whole length.Preferably, said expression enhancing element comprises or consists ofany of the sequences selected from the group consisting of SEQ ID NO:59-61. Most preferably, said expression enhancing element comprises orconsists of a sequence that is represented by SEQ ID NO: 59.

Eleventh Aspect

In an eleventh aspect, the present invention provides a nucleic acidconstruct comprising a nucleic acid molecule according to the tenthaspect of the invention. A nucleic acid construct of the inventioncomprises an expression enhancing element according to the tenth aspectof the present invention. Preferably, said nucleic acid construct is arecombinant and/or isolated construct as defined herein. Preferably,said nucleic acid construct further comprises a heterologous promoter,wherein preferably said expression enhancing element and saidheterologous promoter are configured to be both operably linked to anoptional nucleotide sequence of interest as defined herein below.“Heterologous promoter” is to be understood herein as a promoter that isnot naturally operably linked to the expression enhancing element of theinvention, i.e. a contiguous sequence comprising said expressionenhancing element and said heterologous promoter does not occur innature as neighboring sequences but can be synthesized as a recombinantsequence.

Preferably within this aspect, said heterologous promoter is locatedwithin a nucleic acid construct of the invention downstream or at the 3′site of the expression enhancing element of the invention. Preferably,said heterologous promoter is located within a construct of theinvention upstream or at the 5′ site of the nucleotide sequence of theinvention encoding a protein or polypeptide of interest. In anembodiment of the invention the heterologous promoter is a promotercapable of initiating transcription in the host cell of choice.Heterologous promoters as used herein include tissue-specific,tissue-preferred, cell-type specific, inducible and constitutivepromoters as defined herein. Heterologous promoters and/or regulatingsequences that may be employed in expression of polypeptides accordingto the present invention, preferably in mammalian cells, include, butare not limited to, the human or murine cytomegalovirus (CMV) promoter,a simian virus (SV40) promoter, a human or mouse ubiquitin C promoter, ahuman or mouse or rat elongation factor alpha (EF1-a) promoter, mouse orhamster beta-actin promoter, or a hamster rpS21 promoter. The Tet-Offand Tet-On elements upstream of a minimal promoter such as a CMVpromoter forms an example of an inducible mammalian promoter. Examplesof suitable yeast and fungal promoters are Leu2 promoter, the galactose(Gal1 or Gal 7) promoter, alcohol dehydrogenase I (ADH1) promoter,glucoamylase (Gla) promoter, triose phosphate isomerase (TPI) promoter,translational elongation factor EF-I alpha (TEF2) promoter,glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter, alcoholoxidase (AOX1) promoter, or glutamate dehydrogenase (gdhA) promoter. Anexample of a strong ubiquitous promoter for expression in plants iscauliflower mosaic virus (CaMV) 35S promoter. Preferably, the nucleicacid construct of the invention comprises a heterologous promoter thatis represented by a sequence that has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%to SEQ ID NO: 58. More preferably, the nucleic acid construct of thisaspect of the invention comprises a heterologous promoter that isrepresented by a sequence that has at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%to SEQ ID NO: 57.

In a further preferred embodiment within this aspect, the nucleic acidconstruct of the invention further comprises one or more additionalexpression regulating elements, wherein preferably said expressionenhancing element, said heterologous promoter and said one or moreadditional expression regulating elements are configured to be alloperably linked to an optional nucleotide sequence of interest asdefined herein below. An “additional expression regulating element” isto be understood herein as an element in addition to the expressionenhancing element and/or promoter as defined herein above which may bean additional expression enhancing element or a distinct expressionenhancing element or an expression regulating element in its broadestsense. An additional expression regulating element as encompassed by thepresent invention can be involved in the transcriptional and/ortranslational regulation of a gene, including but not limited to,5′-UTR, 3′-UTR, enhancer, promoter, intronic sequence, polyadenylationsignal and chromatin control elements such as scaffold/matrix attachmentregions, ubiquitous chromatin opening element, cytosine phosphodiesterguanine pairs and stabilizing and anti-repressor elements, and anyderivatives thereof. Other optional regulating elements that may bepresent in the nucleic acid construct of the invention include, but arenot limited to, coding nucleotide sequences of homologous and/orheterologous nucleotide sequences, including the Iron Responsive Element(IRE), Translational cis-Regulatory Element (TLRE) or uORFs in 5′ UTRsand poly(U) stretches in 3′ UTRs.

Preferably within this aspect, said additional expression regulatingelement comprises or consists of an intronic sequence as defined herein.Preferably, the intronic sequence encompassed within the additionalexpression regulating element comprises at least of an acceptor splicesite which is understood herein as to comprise the splice site AGpreferably preceded by a polypyrimidine tract nucleotide sequence,optionally separated from splice site AG by 1-50 nucleotides, andoptionally further comprising a branch site comprising the sequenceY-T-N-A-Y, at the 5′ site of the polypyrimidine tract nucleotidesequence, wherein the branch site may have the nucleotide sequenceC-Y-G-A-C. An acceptor splice site is understood herein as a splice sitethat, when combined with a donor splice site encompassed within aconstruct, results in the formation of an intron as defined in theDefinition section. Preferably, the acceptor splice site or splice siteAG has the sequence [Y-rich]-N-Y-A-G-[cut]. Preferably, the acceptorsplice site or splice site AG has the sequence [Y-rich]-N-Y-A-G-[cut]-Rin case the host cell is a mammalian cell, [Y-rich]-D-Y-A-G-[cut]-R or[Y-rich]-D-Y-A-G-[cut]-R-W in case the host cell is a plant cell,[Y-rich]-A-Y-A-G-[cut] in case the host cell is a yeast cell and[Y-rich]-N-Y-A-G-[cut] in case the host cell is an insect cell.“[Y-rich]” is to be understood herein as a polypyrimidine tract which ispreferably defined as a consecutive sequence of at least 10 nucleotidescomprising at least 6, 7, 8, 9 or preferably 10 pyrimidine nucleotides.Preferably, said acceptor splice site or splice site GT has the sequenceY-A-G-[cut]-R. Preferably, said intronic sequence encompassed withinsaid additional expression regulating element consists of said acceptorsplice site or splice site AG. The intronic sequence preferablycomprises or consists of a nucleotide sequence having at least 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tonucleotides 171-277 of SEQ ID NO: 14, nucleotides 171-274 of SEQ ID NO:19, nucleotides 133-210 of SEQ ID NO: 20, nucleotides 134-211 of SEQ IDNO: 21, nucleotides 134-226 of SEQ ID NO: 22, nucleotides 134-226 of SEQID NO: 23, nucleotides 133-225 of SEQ ID NO: 24, nucleotides 134-226 ofSEQ ID NO: 25, nucleotides 146-257 of SEQ ID NO: 26, or nucleotides147-223 of SEQ ID NO: 27, or nucleotides 970-1449 of SEQ ID NO: 1 ornucleotides 667-1228 of SEQ ID NO: 2. Preferably, said intronic sequencecomprised within said additional expression regulating element furthercomprises a donor splice site as defined herein. Even more preferred,said intronic sequence encompassed within the additional expressionregulating element is a intron as defined in the Definition section.Most preferably, the intronic sequence encompassed within the additionalexpression regulating element comprises or consists of a nucleotidesequence having at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to nucleotides 171-277 of SEQ ID NO:14, nucleotides 171-274 of SEQ ID NO: 19, nucleotides 133-210 of SEQ IDNO: 20, nucleotides 134-211 of SEQ ID NO: 21, nucleotides 134-226 of SEQID NO: 22, nucleotides 134-226 of SEQ ID NO: 23, nucleotides 133-225 ofSEQ ID NO: 24, nucleotides 134-226 of SEQ ID NO: 25, nucleotides 146-257of SEQ ID NO: 26, or nucleotides 147-223 of SEQ ID NO: 27.

Also preferred within this aspect is an expression regulating elementthat is a translation enhancing element. Preferably, a translationenhancing element allows for an increase in protein or polypeptideexpression of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared toexpression of said protein or polypeptide using a construct which onlydiffers in that it is free of said translation enhancing element,preferably when tested in a system as exemplified in the Examples whichare enclosed herein. More specifically, preferably the expression ofnucleotide sequence of interest encoding for secreted alkalinephosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising atranslation enhancing element to be tested and a CMV promoterrepresented by SEQ ID NO: 57, operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said nucleotide sequence ofinterest which are measured under the same conditions except that theexpression vector is free of said translation enhancing element to betested.

Preferably within this aspect, said translation enhancing elementcomprises or consists of a nucleotide sequence that has at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to SEQ ID NO: 3-51 over its whole length.Preferably, said translation enhancing element comprises or consists ofa nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100identity to SEQ ID NO: 19 over its whole length. More preferably, saidtranslation enhancing element comprises or consists of a nucleotidesequence that has at least 90% identity to SEQ ID NO: 3-51 over itswhole length. Also preferred within this aspect is a translationenhancing element that comprises or consists of a nucleotide sequencethat comprises:

-   -   i) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides;    -   ii) a TC-rich nucleotide sequence comprising at least 8        consecutive C or T nucleotides, at least 3 A-rich nucleotide        sequences comprising at least 5 consecutive A nucleotides, and a        GT-rich nucleotide sequence comprising at least 10 nucleotides,        at least 80% of which are G or T nucleotides, said expression        enhancing element not comprising a GAA repeat nucleotide        sequence; or,    -   iii) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides, wherein said GAA repeat nucleotide sequence is        located 3′ of any one or more of said TC-rich nucleotide        sequence, A-rich nucleotide sequences, and/or GT-rich nucleotide        sequence.

The GAA repeat nucleotide sequence, the TC-rich nucleotide sequence, theA-rich nucleotide sequence, the GT-rich nucleotide sequence have alreadybeen defined herein in the first aspect of the invention. Thesedefinitions also applied here.

Preferably within said aspect, said additional expression regulatingelement comprises a translation enhancing element as defined herein andan intronic sequence.

Preferably within said aspect, said additional expression regulatingelement is located within a nucleic acid construct of the inventiondownstream or the 3′ site of a heterologous promoter. Preferably, saidadditional expression regulating element is located within a nucleicacid construct of the invention upstream or at the 5′ site of a nucleicacid sequence encoding a protein or polypeptide of interest. Moreover,preferably a nucleic acid construct of the invention comprises thefollowing nucleotide sequences indicated here in their relativepositions in the 5′ to 3′ direction: (i) an expression enhancingelement, (ii) a heterologous promoter, optionally (iii) an additionalexpression regulating element, and optionally (iv) a nucleotide sequenceof interest, wherein preferably said expression enhancing element, saidheterologous promoter and said additional expression regulating elementare configured to be all operably linked to said optional nucleotidesequence of interest as defined herein below. It is to be understoodthat said expression enhancing element, said heterologous promoter, andoptionally said additional expression regulating element of the nucleicacid construct of the invention are all configured to be operably linkedto the same, single nucleotide sequence of interest.

The inventors found an unexpected synergistic effect when the expressionenhancing element of the invention is combined with an additionalexpression regulating element as defined herein in an expressionconstruct for expressing a protein or polypeptide of interest. In astably transfected pool with both an expression enhancing element and anadditional expression regulating element, the protein yield wassignificantly higher than the yield expected based on addition of theseparate effects of either element. Preferably, said increase of proteinor polypeptide expression is copy number independent as established byan assay suitable to determine copy number dependency by a skilledperson, such as, but not limit to, a triplex Taqman assay as furtherdetailed in Example 4 of the present invention. Preferably, said nucleicacid construct is a recombinant and/or isolated construct as definedherein. Preferably, said nucleic acid construct further comprises anucleotide sequence of interest operably linked to and/or under thecontrol of said expression enhancing element, said heterologous promoterand optionally said additional expression regulating element as definedherein. The presence of a nucleotide sequence of interest is optional.“Optional” is to be understood herein as not necessarily being presentin an expression construct. For instance, such nucleotide sequence ofinterest need not be present in a commercialized expression vector, butmay be readily introduced by a person skilled in the art before use in amethod of the invention. It is to be understood that said expressionenhancing element, said heterologous promoter, and optionally saidadditional expression regulating element are all configured to beoperably linked to the same, single nucleotide sequence of interest.Preferably, said nucleic acid construct of the tenth aspect of theinvention has at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to SEQ ID NO 73, 74, 75 or 76.

In a preferred embodiment within this aspect, said nucleotide sequenceof interest is a nucleotide sequence encoding a protein or polypeptideof interest. The protein or polypeptide of interest can be a homologousprotein or polypeptide, but in a preferred embodiment of the inventionthe protein or polypeptide of interest is a heterologous protein orpolypeptide. A nucleotide sequence encoding a heterologous protein orpolypeptide may be derived in whole or in part from any source known tothe art, including a bacterial or viral genome or episome, eukaryoticnuclear or plasmid DNA, cDNA or chemically synthesised DNA. Thenucleotide sequence encoding a protein or polypeptide of interest mayconstitute an uninterrupted coding region or it may include one or moreintrons bounded by appropriate splice junctions. It can further becomposed of segments derived from different sources, naturally occurringor synthetic. The nucleotide sequence encoding the protein orpolypeptide of interest according to the method of the invention ispreferably a full-length nucleotide sequence, but can also be afunctionally active part or other part of said full-length nucleotidesequence. The nucleotide sequence encoding the protein or polypeptide ofinterest may also comprise signal sequences directing the protein orpolypeptide of interest when expressed to a specific location in thecell or tissue. Furthermore, the nucleotide sequence encoding theprotein or polypeptide of interest can also comprise sequences whichfacilitate protein purification and protein detection by for instanceWestern blotting and ELISA (e.g. c-myc or polyhistidine sequences).

The protein or polypeptide of interest in this aspect has already beendefined earlier herein in the first aspect of the invention.

In an alternative embodiment, said nucleotide sequence of interest isnot a coding sequence for a protein or a polypeptide but may be afunctional nucleotide sequence. This alternative embodiment of thisaspect has already been defined earlier herein in the first aspect ofthe invention.

Twelfth Aspect

In a twelfth aspect, the present invention provides an expression vectorcomprising a nucleic acid construct according to the eleventh aspect ofthe invention. The expression vector of the invention preferably is aplasmid, cosmid or phage or nucleotide sequence, linear or circular, ofa single or double stranded DNA or RNA, derived from any source, inwhich a number of nucleotide sequences have been joined or recombinedinto a unique construction which is capable of introducing any one ofthe nucleotide sequences of the invention in sense or antisenseorientation into a cell. The choice of vector is dependent on therecombinant procedures followed and the host cell used. The vector maybe an autonomously replicating vector or may replicate together with thechromosome into which it has been integrated. Preferably, the vectorcontains a selection marker. Useful markers are dependent on the hostcell of choice and are well known to persons skilled in the art and areselected from, but not limited to, the selection markers as defined inthird aspect of the invention. A preferred expression vector is thepcDNA3.1 expression vector. Preferred selection markers are the neomycinresistance gene, zeocin resistance gene and blasicidin resistance gene.

Thirteenth Aspect

In a thirteenth aspect, the present invention provides a cell comprisinga nucleic acid molecule according to the tenth aspect of the invention,and/or a nucleic acid construct according to the eleventh aspect of theinvention, and/or an expression vector according to the twelfth aspectof the invention as defined herein. The type of cell within the contextof this aspect is the same as the one defined in the context of thethird aspect. Therefore, another aspect of the invention relates to ahost cell that is genetically modified, preferably by a method of theinvention, in that a host cell comprises a nucleic acid construct asdefined above in the thirteenth aspect. For transformation procedures inplants, suitable bacteria include Agrobacterium tumefaciens andAgrobacterium rhizogenes.

A nucleic acid construct within the context of this thirteenth aspect isas the one of the third aspect: it is preferably stably maintained,either as an autonomously replicating element, or, more preferably, thenucleic acid construct is integrated into the host cell's genome, inwhich case the construct is usually integrated at random positions inthe host cell's genome, for instance by non-homologous recombination.Stably transformed host cells are produced by known methods. Thedefinition of the term stable transformation and methods encompassed forstable transformation have already been provided under the third aspect.

Alternatively, a protein or polypeptide of interest may be expressed ina host cell, e.g., a mammalian cell, relying on transient expressionfrom vectors.

A nucleic acid construct according to this aspect preferably alsocomprises a marker gene which can provide selection or screeningcapability in a treated host cell.

All definitions relating to selectable markers and types of selectablemarkers including the example of the use of the luciferase gene asselectable marker, the example of a first category of marker based on acell's metabolism and the use of a mutant cell line which lacks theability to grow independent of a supplemented media, the example ofdominant selection have already been provided in the third aspect. Theyalso apply here in the thirteenth aspect of the invention.

When a transformed host cell is obtained with a method according to theinvention (see below), a host tissue may be regenerated from saidtransformed cell in a suitable medium, which optionally may containantibiotics or biocides known in the art for the selection oftransformed cells.

Resulting transformed host tissues are preferably identified by means ofselection using a selection marker gene as present on a nucleic acidconstruct as defined herein.

Fourteenth Aspect

In a fourteenth aspect, the present invention provides a method forexpressing and optionally purifying a protein or polypeptide of interestcomprising the step of:

-   -   a. providing a nucleic acid construct according to the eleventh        aspect of the invention comprising a nucleotide sequence        encoding a protein or polypeptide of interest; and,    -   b. contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c. allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,    -   d. purifying said protein or polypeptide of interest.

In a preferred embodiment of the method according to the invention, anucleic acid construct as defined above in the eleventh aspect of theinvention is used. The method of the invention may be an in vitro or exvivo method. The method of the invention may be applied on a cellculture, organism culture, or tissue culture. Alternatively, next to theexpression in host cells the protein or polypeptide of interest can beproduced in cell-free translation systems using RNAs derived from thenucleic acid constructs of the present invention. The method of theinvention may be performed on cultured cells.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to express the protein orpolypeptide of interest, and optionally said protein or polypeptide issubsequently recovered. For example, the transformed cell may besubjected to conditions leading to expression of the protein orpolypeptide of interest. The person skilled in the art is well aware oftechniques to be used for expressing or overexpressing the protein orpolypeptide of interest. Methods in which the transformed cell does notneed to be subjected to specific conditions leading to expression of theprotein or polypeptide of interest, but in which the protein orpolypeptide of interest is automatically (e.g., constitutively)expressed, are also included in the method of the present invention.

Purification steps and definitions related to these steps as thedefinition of an isolated protein or polypeptide are the same as in themethod of the fourth aspect and have been earlier defined herein. Ifdesired as defined in the method of the fourth aspect, the nucleotidesequence encoding a protein or polypeptide of interest may be ligated toa heterologous nucleotide sequence to encode a fusion protein orpolypeptide to facilitate protein purification and protein detection onfor instance Western blot and in an ELISA. Suitable heterologoussequences include, but are not limited to, the nucleotide sequencescoding for proteins such as for instance glutathione-S-transferase,maltose binding protein, metal-binding polyhistidine, green fluorescentprotein, luciferase and beta-galactosidase. The protein or polypeptidemay also be coupled to non-peptide carriers, tags or labels thatfacilitate tracing of the protein or polypeptide, both in vivo and invitro, and allow for the identification and quantification of binding ofthe protein or polypeptide to substrates. Such labels, tags or carriersare well-known in the art and include, but are not limited to, biotin,radioactive labels and fluorescent labels.

Preferably, the method of this fourteenth aspect of the invention allowsfor an increase in expression of a protein or polypeptide of interest.Preferably, expression levels are established in an expression systemusing an expression construct according to the eleventh aspect of theinvention comprising an expression enhancing element and a heterologouspromoter operably linked to a nucleotide sequence encoding a protein orpolypeptide of interest according to the eleventh aspect of theinvention. Preferably, said protein or polypeptide of interest is asecreted protein or polypeptide and expression of said protein orpolypeptide of interest is detected by a suitable assay such as an ELISAassay, Western blotting or, dependent on the identity of the protein orpolypeptide of interest, any suitable protein identification and/orquantification assay known to the person skilled in the art. Preferably,the method of the invention allows for an increase in protein orpolypeptide expression of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% ascompared to a method which only differs in that a construct is used instep a) that it is free of said expression enhancing element, preferablywhen tested in a system as exemplified in the Examples which areenclosed herein. More specifically, preferably the expression ofnucleotide sequence of interest encoding for secreted alkalinephosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprisingan expression enhancing element to be tested and a CMV promoterrepresented by SEQ ID NO: 57, operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said nucleotide sequence ofinterest which are measured under the same conditions except that theexpression vector is free of said expression enhancing element to betested. Preferably, said increase of protein or polypeptide expressionis copy number independent as established by an assay suitable todetermine copy number dependency by a skilled person such as, but notlimit to, a triplex Taqman assay as further detailed in Example 4 of thepresent invention.

Fifteenth Aspect

In a fifteenth aspect, the present invention provides a method forexpressing a protein or polypeptide of interest in an organism,comprising the steps of:

-   -   a) providing a nucleic acid construct according to the eleventh        aspect comprising a nucleotide sequence encoding a protein or        polypeptide of interest of the invention; and,    -   b) contacting a target cell and/or target tissue of an organism,        with said nucleic acid construct to obtain a transformed target        cell and/or transformed target tissue, allowing said transformed        cell to express the protein or polypeptide of interest; and        optionally,    -   c) allowing said transformed target cell to develop into a        transformed organism; and, optionally,    -   d) allowing said transformed organism to express the protein or        polypeptide of interest, for example, subjecting said        transformed organism to conditions leading to expression of the        protein or polypeptide of interest, and optionally recovering        said protein or polypeptide.

The target cell may be an embryonal target cell, e.g., embryonic stemcell, for example, derived from a non-human mammalian, such as bovine,porcine, et cetera species. Preferably, said target cell is not a humanembryonic stem cell. In the case of a multicellular fungus, such targetcell may be a fungal cell that can be proliferated into saidmulticellular fungus. When a transformed plant tissue or plant cell(e.g., pieces of leaf, stem segments, roots, but also protoplasts orplant cells cultivated by suspension) is obtained with this methodaccording to the invention, whole plants can be regenerated from saidtransformed tissue or cell in a suitable medium, which optionally maycontain antibiotics or biocides known in the art for the selection oftransformed cells. This method of the invention may be applied innucleic acid based vaccination and/or gene therapy preferably in amammal, most preferably in a human. Encompassed within the presentinvention is a method of treatment comprising the method of the presentaspect, wherein the protein or polypeptide of interest is a therapeuticand/or immunogenic protein or polypeptide. The invention also relates toa construct of the eleventh aspect of the invention for treatment,wherein the protein or polypeptide of interest is a therapeutic and/orimmunogenic protein or polypeptide. Furthermore, the invention relatesto the use of a construct of the eleventh aspect of the invention forthe manufacture of a medicament, wherein the protein or polypeptide ofinterest is a therapeutic and/or immunogenic protein or polypeptide.

Furthermore, an embodiment of the invention is a non-human transformedorganism. Said organism is transformed with a nucleotide sequence,recombinant nucleic acid construct, or vector according to the presentinvention, and is capable of producing the polypeptide of interest. Thisincludes a non-human transgenic organism, such as a transgenic non-humanmammalian, transgenic plant (including propagation, harvest and tissuematerial of said transgenic plant, including, but not limited to, leafs,roots, shoots and flowers), multicellular fungus, and the like.

Preferably, the method of this aspect of the invention allows for anincrease in expression of a protein or polypeptide of interest in saidorganism or at least in one tissue or organelle or organ of saidorganism. Preferably, expression levels are established in an expressionsystem using an expression construct according to the eleventh aspect ofthe invention comprising an expression enhancing element and aheterologous promoter operably linked to a nucleotide sequence encodinga protein or polypeptide of interest according to the eleventh aspect ofthe invention. Preferably, said protein or polypeptide of interest is asecreted protein or polypeptide and expression of said protein orpolypeptide of interest is detected by a suitable assay such as an ELISAassay, Western blotting or, dependent on the identity of the protein orpolypeptide of interest, any suitable protein identification and/orquantification assay known to the person skilled in the art. Preferably,this method of the invention allows for an increase in protein orpolypeptide expression of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% insaid organism or at least in one tissue or organelle or organ of saidorganism. as compared to a method which only differs in that a constructis used in step a) that it is free of said expression enhancing element,preferably when tested in a system as exemplified in the Examples whichare enclosed herein. More specifically, preferably the expression ofnucleotide sequence of interest encoding for secreted alkalinephosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprisingan expression enhancing element to be tested and a CMV promoterrepresented by SEQ ID NO: 57, operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said nucleotide sequence ofinterest which are measured under the same conditions except that theexpression vector is free of said expression enhancing element to betested.

Preferably, said increase of protein or polypeptide expression is copynumber independent as established by an assay suitable to determine copynumber dependency by a skilled person such as, but not limit to, atriplex Taqman assay as further detailed in Example 4 of the presentinvention.

Sixteenth Aspect

In a sixteenth aspect, the present invention provides a method fortranscription and optionally purifying the produced transcriptcomprising the step of:

-   -   a) providing a nucleic acid construct according to the eleventh        aspect comprising a nucleotide sequence of interest of the        invention; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

In a preferred embodiment of this method according to the invention anucleic acid construct as defined above in the eleventh aspect is used.The method of the invention may be an in vitro or ex vivo method. Themethod of the invention may be applied on a cell culture, organismculture, or tissue culture. The method of the invention may be appliedin nucleic acid based vaccination and/or gene therapy preferably in amammal, preferably in a human. Encompassed within the present inventionis a method for treatment comprising or consisting of the method of thepresent aspect, wherein the nucleotide sequence of interest encodes fora therapeutic transcript. The invention also relates to a construct ofthe eleventh aspect of the invention for use in treatment, wherein thenucleotide sequence of interest encodes for a therapeutic transcript.Furthermore, the invention relates to the use of a construct of theeleventh aspect of the invention for the manufacture of a medicament,wherein the nucleotide sequence of interest encodes for a therapeutictranscript.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to produce a transcript ofthe nucleotide sequence of interest, and optionally the producedtranscript is subsequently recovered. For example, the transformed cellmay be subjected to conditions leading to transcription the nucleotidesequence of interest. The person skilled in the art is well aware oftechniques to be used for transcription the nucleotide sequence ofinterest. Methods in which the transformed cell does not need to besubjected to specific conditions leading to transcription of thenucleotide sequence of interest, but in which the nucleotide sequence ofinterest is automatically (e.g., constitutively) transcribed, are alsoincluded in the method of the present invention.

Purification steps depend on the transcript produced. The term“isolation” indicates that the transcript is found in a condition otherthan its native environment. In a preferred form, the isolatedtranscript is substantially free of other cellular components,particularly other homologous cellular components such as homologousproteins. It is preferred to provide the transcript in a greater than40% pure form, more preferably greater than 60% pure form. Even morepreferably it is preferred to provide the transcript in a highlypurified form, i.e., greater than 80% pure, more preferably greater than95% pure, and even more preferably greater than 99% pure, as determinedby Northern blot.

Preferably, the method of this aspect of the invention allows for anincrease in transcription of a nucleotide sequence of interest.Preferably, transcription levels are established in an expression systemusing an expression construct according to the second aspect of theinvention comprising an expression enhancing element and a heterologouspromoter operably linked to a nucleotide sequence of interest accordingto the second aspect of the invention. Preferably, transcription of saidnucleotide sequence of interest is detected by a suitable assay such asRT-qPCR. Preferably, the method of the invention allows for an increasein transcription of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared to amethod which only differs in that a construct is used in step a) that itis free of said expression enhancing element, preferably when tested ina system as exemplified in the Examples which are enclosed herein. Morespecifically, preferably the transcription of a nucleotide sequence ofinterest encoding for secreted alkaline phosphatase (SeAP) is measuredin a mammalian cell system, most preferably in CHO cells, using apcDNA3.1 expression vector comprising an expression enhancing element tobe tested and a CMV promoter represented by SEQ ID NO: 57, operablylinked to said nucleotide sequence of interest. Transcription ispreferably measured using RT-qPCR and transcription levels are comparedto transcription levels of said nucleotide sequence of interest measuredunder the same conditions except that the expression vector used is freeof said expression enhancing element to be tested.

Seventeenth Aspect

In an seventeenth aspect, the present invention provides a use of anucleic acid molecule according to the tenth aspect of the invention,and/or a use of a nucleic acid construct according to the eleventhaspect of the invention, and/or a use of an expression vector accordingto the twelfth aspect of the invention, and/or a use of a cell accordingto the thirteenth aspect of the invention, for the transcription of anucleotide sequence of interest and/or the expression of a protein orpolypeptide of interest.

Eighteenth Aspect

In a eighteenth aspect, the present invention provides for a nucleicacid molecule according to according to the tenth aspect of theinvention, and/or a nucleic acid construct according to the eleventhaspect of the invention, and/or an expression vector according to thetwelfth aspect of the invention, and/or a cell according to thethirteenth aspect of the invention for use as a medicament. Theinvention also relates to a method of treatment comprising theadministration of a nucleic acid molecule according to the tenth aspectof the invention, and/or a nucleic acid construct according to theeleventh aspect of the invention, and/or an expression vector accordingto the twelfth aspect of the invention, and/or a cell according to thethirteenth aspect of the invention, wherein preferably saidadministration is to a mammal, more preferably to a human. Preferably,said treatment is nucleic acid based vaccination and/or gene therapypreferably in a mammal, most preferably in a human. Furthermore, theinvention relates to the use of a nucleic acid molecule according toaccording to the tenth aspect of the invention, and/or the use of anucleic acid construct according to the eleventh aspect of theinvention, and/or the use of an expression vector according to thetwelfth aspect of the invention, and/or the use of a cell according tothe thirteenth aspect of the invention, for the preparation of amedicament. Preferably said medicament is for nucleic acid basedvaccination and/or gene therapy preferably in a mammal, most preferablyin a human.

Nineteenth Aspect

In a nineteenth aspect, the present invention provides a nucleic acidmolecule that is represented by a nucleotide sequence that has at least50% identity with SEQ ID NO: 88 for increasing transcription of anucleotide sequence of interest and/or expression of a protein orpolypeptide of interest. Within the context of the nineteenth to twentyseventh aspect, said identity percentage is preferably assessed over thewhole length of SEQ ID NO:88. However, it is not excluded that saididentity percentage is assessed over part of SEQ ID NO:88 as defined inthe section entitled definitions. Preferably, said nucleotide sequenceof the invention is capable of increasing the transcription of anucleotide sequence of interest and/or expression of a protein orpolypeptide of interest. Said nucleic acid molecule represented by anucleotide sequence that has at least 50% identity with SEQ ID NO:88 maybe called a transcription regulating sequence.

Preferably within this aspect, transcription levels are established inan expression system using an expression construct comprising saidnucleotide sequence operably linked to a nucleotide sequence of interestusing a suitable assay such a RT-qPCR. Preferably, the nucleotidesequence of the invention allows for an increase in transcription of atleast 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 500%, 1000%, 1500% or 2000% as compared to transcription levelsusing a construct wherein the nucleotide sequence having at least 50%identity with SEQ ID NO:88 has been replaced by an alternative sequence,preferably as exemplified in example 11 which is enclosed herein. Morespecifically, preferably the transcription of a nucleotide sequence ofinterest encoding for secreted alkaline phosphatase (SeAP) is measuredin a mammalian cell system, most preferably in CHO cells, using apcDNA3.1 expression vector comprising the nucleotide sequence of theinvention operably linked to said nucleotide sequence of interest.Transcription is preferably measured using RT-qPCR and transcriptionlevels are compared to transcription levels of said nucleotide sequenceof interest measured under the same conditions except that in theexpression vector used the nucleotide sequence having at least 50%identity with SEQ ID NO:88 has been replaced by an alternative sequence,preferably as exemplified in example 11 which is enclosed herein.

Preferably within this aspect, expression levels are established in anexpression system using an expression construct comprising saidnucleotide sequence having at least 50% identity with SEQ ID NO:88 andwhich is operably linked to a nucleotide sequence encoding a protein orpolypeptide of interest. Preferably, said protein or polypeptide ofinterest is a secreted protein or polypeptide and expression of saidprotein or polypeptide of interest is detected by a suitable assay suchas an enzyme-linked immunosorbent assay (ELISA) assay, Western blottingor, dependent on the identity of the protein or polypeptide of interest,any suitable protein identification and/or quantification assay known tothe person skilled in the art. Preferably, the nucleotide sequencehaving at least 50% identity with SEQ ID NO:88 allows for an increase inprotein or polypeptide expression of at least 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%, 1500% or2000% as compared to expression of said protein or polypeptide using aconstruct which only differs in that said nucleotide sequence has beenreplaced by an alternative sequence, preferably when tested in a systemas exemplified in example 11 which is enclosed herein. Morespecifically, preferably the expression of a nucleotide sequence ofinterest encoding for secreted alkaline phosphatase (SeAP) is measuredin a mammalian cell system, most preferably in CHO cells, using apcDNA3.1 expression vector comprising a nucleotide sequence having atleast 50% identity with SEQ ID NO:88 operably linked to said nucleotidesequence of interest. Expression is preferably measured by measuring theconversion of any suitable alkaline phosphatase substrate and expressionlevels are compared to expression levels of said isolated nucleic acidmolecule as defined herein. In a preferred embodiment, said nucleotidesequence having at least 50% identity with SEQ ID NO:88 is a sequencethat is derived from the UBC ubiquitin gene. Preferably, said nucleotidesequence is derived from a mammalian UBC ubiquitin gene. Morepreferably, said nucleotide sequence is derived from the Cricetulusgriseus homologous gene of the human UBC ubiquitin gene, said gene beingindicated as the Cricetulus sp. gene for polyubiquitin, or CRUPUQ(GenBank D63782).

In a preferred embodiment, said nucleotide sequence comprises orconsists of a sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity with SEQ ID NO: 88 over its whole length. Preferably, saidsequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ IDNO: 88 over its whole length comprises a promoter as defined in theDefinition section. Preferably, said promoter is capable of initiatingtranscription of a nucleotide sequence of interest and/or expression ofa polypeptide or protein or polypeptide of interest encoded by anucleotide sequence in a host cell as defined herein below. Preferably,said nucleotide sequence for increasing expression is a contiguoussequence of at least 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600 or 2617 in length, preferably at least 2617nucleotides in length of SEQ ID NO: 88. Preferably, said nucleotidesequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to SEQ ID NO: 88 is at most 8000nucleotides in length. Preferably, said nucleotide sequence is at most8000, 7000, 6000, 5000, 4000, 3000, 2617 in length. Most preferably,said sequence being 2617 nucleotides in length. Preferably, nucleotidesequence is at most 8000, 7000, 6000, 5000, 4000, 3000, 2617 nucleotidesin length and has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% identity to SEQ ID NO: 88 over its whole length.

Twentieth Aspect

In a twentieth aspect, the present invention provides a nucleic acidconstruct comprising a nucleic acid molecule according to the nineteenthaspect of the invention. A nucleic acid construct of the inventioncomprises a nucleotide sequence according to the nineteenth aspect ofthe present invention. Preferably, said nucleic acid construct is arecombinant and/or isolated construct as defined herein. Preferably,said nucleic acid construct further comprises an optional nucleotidesequence of interest as defined herein below wherein the nucleotidesequence of the invention is operably linked to said optional nucleicacid sequence of interest.

In a further preferred embodiment within this aspect, the nucleic acidconstruct of the invention further comprises one or more additionalexpression regulating elements, wherein preferably said nucleotidesequence and said one or more additional expression regulating elementsare configured to be all operably linked to an optional nucleotidesequence of interest as defined herein below. An “additional expressionregulating element” is to be understood herein as an element in additionto the nucleotide sequence as defined herein above which may be anadditional expression regulating element or a distinct expressionregulating element or an additional expression enhancing element or adistinct expression enhancing element. An additional expressionregulating element as encompassed by the present invention can beinvolved in the transcriptional and/or translational regulation of agene, including but not limited to, 5′-UTR, 3′-UTR, enhancer, promoter,intron, polyadenylation signal and chromatin control elements such asscaffold/matrix attachment regions, ubiquitous chromatin openingelement, cytosine phosphodiester guanine pairs and stabilizing andanti-repressor elements, and any derivatives thereof. Other optionalregulating elements that may be present in the nucleic acid construct ofthe invention include, but are not limited to, coding nucleotidesequences of homologous and/or heterologous nucleotide sequences,including the Iron Responsive Element (IRE), Translationalcis-Regulatory Element (TLRE) or uORFs in 5′ UTRs and poly(U) stretchesin 3′ UTRs.

Also preferred within this aspect is an expression regulating elementthat is a translation enhancing element. Preferably, a translationenhancing element allows for an increase in protein or polypeptideexpression of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 500%, 1000%, 1500% or 2000% as compared toexpression of said protein or polypeptide using a construct which onlydiffers in that it is free of said translation enhancing element,preferably when tested in a system as exemplified in the Examples whichare enclosed herein. More specifically, preferably the expression ofnucleotide sequence of interest encoding for secreted alkalinephosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising atranslation enhancing element to be tested and a nucleotide sequencehaving at least 50% identity with SEQ ID NO: 88, operably linked to saidnucleotide sequence of interest. Expression is preferably measured bymeasuring the conversion of any suitable alkaline phosphatase substrateand expression levels are compared to expression levels of saidnucleotide sequence of interest which are measured under the sameconditions except that the expression vector is free of said translationenhancing element to be tested.

Preferably within this aspect, said translation enhancing elementcomprises or consists of a nucleotide sequence that has at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identity to SEQ ID NO: 3-51 over its whole length.Preferably, said translation enhancing element comprises or consists ofa nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100identity to SEQ ID NO: 19 over its whole length. More preferably, saidtranslation enhancing element comprises or consists of a nucleotidesequence that has at least 90% identity to SEQ ID NO: 3-51 over itswhole length. Also preferred within this aspect is a translationenhancing element that comprises or consists of a nucleotide sequencethat comprises:

-   -   i) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides;    -   ii) a TC-rich nucleotide sequence comprising at least 8        consecutive C or T nucleotides, at least 3 A-rich nucleotide        sequences comprising at least 5 consecutive A nucleotides, and a        GT-rich nucleotide sequence comprising at least 10 nucleotides,        at least 80% of which are G or T nucleotides, said expression        enhancing element not comprising a GAA repeat nucleotide        sequence; or,    -   iii) a GAA repeat nucleotide sequence, a TC-rich nucleotide        sequence comprising at least 8 consecutive C or T nucleotides,        at least 3 A-rich nucleotide sequences comprising at least 5        consecutive A nucleotides, a GT-rich nucleotide sequence        comprising at least 10 nucleotides, at least 80% of which are G        or T nucleotides, wherein said GAA repeat nucleotide sequence is        located 3′ of any one or more of said TC-rich nucleotide        sequence, A-rich nucleotide sequences, and/or GT-rich nucleotide        sequence.

The GAA repeat nucleotide sequence, the TC-rich nucleotide sequence, theA-rich nucleotide sequence, the GT-rich nucleotide sequence have alreadybeen defined herein in the first aspect of the invention. Thesedefinitions also applied here.

Preferably within said aspect, said additional expression regulatingelement is located within a nucleic acid construct of the inventionhaving at least 50% identity with SEQ ID NO: 88. Preferably, saidadditional expression regulating element is located within a nucleicacid construct of the invention upstream or at the 5′ site of a nucleicacid sequence encoding a protein or polypeptide of interest. Moreover,preferably a nucleic acid construct of the invention comprises thefollowing nucleotide sequences indicated here in their relativepositions in the 5′ to 3′ direction:

-   -   optionally (i) an expression regulating preferably enhancing        element,    -   (ii) a nucleotide sequence having at least 50% identity with SEQ        ID NO:88, optionally (iii) an additional expression regulating        element, and optionally (iv) a nucleotide sequence of interest,        wherein preferably said expression enhancing element, said        nucleotide sequence having at least 50% identity with SEQ ID        NO:88 and said additional expression regulating element are        configured to be all operably linked to said optional nucleotide        sequence of interest as defined herein below. It is to be        understood that said expression enhancing element, said        nucleotide sequence having at least 50% identity with SEQ ID        NO:88, and optionally said additional expression regulating        element of the nucleic acid construct of the invention are all        configured to be operably linked to the same, single nucleotide        sequence of interest.

The presence of a nucleotide sequence of interest is optional.“Optional” is to be understood herein as not necessarily being presentin an expression construct. For instance, such nucleotide sequence ofinterest need not be present in a commercialized expression vector, butmay be readily introduced by a person skilled in the art before use in amethod of the invention. It is to be understood that said expressionenhancing element, said nucleotide sequence having at least 50% identitywith SEQ ID NO:88, and optionally said additional expression regulatingelement are all configured to be operably linked to the same, singlenucleotide sequence of interest.

In a preferred embodiment within this aspect, said nucleotide sequenceof interest is a nucleotide sequence encoding a protein or polypeptideof interest. The protein or polypeptide of interest can be a homologousprotein or polypeptide, but in a preferred embodiment of the inventionthe protein or polypeptide of interest is a heterologous protein orpolypeptide. A nucleotide sequence encoding a heterologous protein orpolypeptide may be derived in whole or in part from any source known tothe art, including a bacterial or viral genome or episome, eukaryoticnuclear or plasmid DNA, cDNA or chemically synthesised DNA. Thenucleotide sequence encoding a protein or polypeptide of interest mayconstitute an uninterrupted coding region or it may include one or moreintrons bounded by appropriate splice junctions. It can further becomposed of segments derived from different sources, naturally occurringor synthetic. The nucleotide sequence encoding the protein orpolypeptide of interest according to the method of the invention ispreferably a full-length nucleotide sequence, but can also be afunctionally active part or other part of said full-length nucleotidesequence. The nucleotide sequence encoding the protein or polypeptide ofinterest may also comprise signal sequences directing the protein orpolypeptide of interest when expressed to a specific location in thecell or tissue. Furthermore, the nucleotide sequence encoding theprotein or polypeptide of interest can also comprise sequences whichfacilitate protein purification and protein detection by for instanceWestern blotting and ELISA (e.g. c-myc or polyhistidine sequences).

The protein or polypeptide of interest in this aspect has already beendefined earlier herein in the first aspect of the invention.

In an alternative embodiment, said nucleotide sequence of interest isnot a coding sequence for a protein or a polypeptide but may be afunctional nucleotide sequence. This alternative embodiment of thisaspect has already been defined earlier herein in the first aspect ofthe invention.

Twenty First Aspect

In a twenty first aspect, the present invention provides an expressionvector comprising a nucleic acid construct according to the twentiethaspect of the invention. The expression vector of the inventionpreferably is a plasmid, cosmid or phage or nucleotide sequence, linearor circular, of a single or double stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a unique construction which is capable of introducingany one of the nucleotide sequences of the invention in sense orantisense orientation into a cell. The choice of vector is dependent onthe recombinant procedures followed and the host cell used. The vectormay be an autonomously replicating vector or may replicate together withthe chromosome into which it has been integrated. Preferably, the vectorcontains a selection marker. Useful markers are dependent on the hostcell of choice and are well known to persons skilled in the art and areselected from, but not limited to, the selection markers as defined inthird aspect of the invention. A preferred expression vector is thepcDNA3.1 expression vector. Preferred selection markers are the neomycinresistance gene, zeocin resistance gene and blasicidin resistance gene.

Twenty Second Aspect

In a twenty second aspect, the present invention provides a cellcomprising a nucleic acid molecule according to the nineteenth aspect ofthe invention, and/or a nucleic acid construct according to thetwentieth aspect of the invention, and/or an expression vector accordingto the twenty first aspect of the invention as defined herein. The typeof cell within the context of this aspect is the same as the one definedin the context of the third aspect.

Therefore, another aspect of the invention relates to a host cell thatis genetically modified, preferably by a method of the invention, inthat a host cell comprises a nucleic acid construct as defined above inthe twentieth aspect. For transformation procedures in plants, suitablebacteria include Agrobacterium tumefaciens and Agrobacterium rhizogenes.

A nucleic acid construct within the context of this twentieth aspect isas the one of the third aspect: it is preferably stably maintained,either as an autonomously replicating element, or, more preferably, thenucleic acid construct is integrated into the host cell's genome, inwhich case the construct is usually integrated at random positions inthe host cell's genome, for instance by non-homologous recombination.Stably transformed host cells are produced by known methods. Thedefinition of the term stable transformation and methods encompassed forstable transformation have already been provided under the third aspect.

Alternatively, a protein or polypeptide of interest may be expressed ina host cell, e.g., a mammalian cell, relying on transient expressionfrom vectors.

A nucleic acid construct according to this aspect preferably alsocomprises a marker gene which can provide selection or screeningcapability in a treated host cell.

All definitions relating to selectable markers and types of selectablemarkers including the example of the use of the luciferase gene asselectable marker, the example of a first category of marker based on acell's metabolism and the use of a mutant cell line which lacks theability to grow independent of a supplemented media, the example ofdominant selection have already been provided in the third aspect. Theyalso apply here in the thirteenth aspect of the invention.

When a transformed host cell is obtained with a method according to theinvention (see below), a host tissue may be regenerated from saidtransformed cell in a suitable medium, which optionally may containantibiotics or biocides known in the art for the selection oftransformed cells.

Resulting transformed host tissues are preferably identified by means ofselection using a selection marker gene as present on a nucleic acidconstruct as defined herein.

Twenty Third Aspect

In a twenty third aspect, the present invention provides a method forexpressing and optionally purifying a protein or polypeptide of interestcomprising the step of:

-   -   a. providing a nucleic acid construct according to the twentieth        aspect of the invention comprising a nucleotide sequence        encoding a protein or polypeptide of interest; and,    -   b. contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c. allowing said transformed cell to express the protein or        polypeptide of interest; and optionally,    -   d. purifying said protein or polypeptide of interest.

In a preferred embodiment of the method according to the invention, anucleic acid construct as defined above in the twentieth aspect of theinvention is used. The method of the invention may be an in vitro or exvivo method. The method of the invention may be applied on a cellculture, organism culture, or tissue culture. Alternatively, next to theexpression in host cells the protein or polypeptide of interest can beproduced in cell-free translation systems using RNAs derived from thenucleic acid constructs of the present invention. The method of theinvention may be performed on cultured cells.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to express the protein orpolypeptide of interest, and optionally said protein or polypeptide issubsequently recovered. For example, the transformed cell may besubjected to conditions leading to expression of the protein orpolypeptide of interest. The person skilled in the art is well aware oftechniques to be used for expressing or overexpressing the protein orpolypeptide of interest. Methods in which the transformed cell does notneed to be subjected to specific conditions leading to expression of theprotein or polypeptide of interest, but in which the protein orpolypeptide of interest is automatically (e.g., constitutively)expressed, are also included in the method of the present invention.

Purification steps and definitions related to these steps as thedefinition of an isolated protein or polypeptide are the same as in themethod of the fourth aspect and have been earlier defined herein. Ifdesired as defined in the method of the fourth aspect, the nucleotidesequence encoding a protein or polypeptide of interest may be ligated toa heterologous nucleotide sequence to encode a fusion protein orpolypeptide to facilitate protein purification and protein detection onfor instance Western blot and in an ELISA. Suitable heterologoussequences include, but are not limited to, the nucleotide sequencescoding for proteins such as for instance glutathione-S-transferase,maltose binding protein, metal-binding polyhistidine, green fluorescentprotein, luciferase and beta-galactosidase. The protein or polypeptidemay also be coupled to non-peptide carriers, tags or labels thatfacilitate tracing of the protein or polypeptide, both in vivo and invitro, and allow for the identification and quantification of binding ofthe protein or polypeptide to substrates. Such labels, tags or carriersare well-known in the art and include, but are not limited to, biotin,radioactive labels and fluorescent labels.

Preferably, the method of this twenty third aspect of the inventionallows for an increase in expression of a protein or polypeptide ofinterest. Preferably, expression levels are established in an expressionsystem using an expression construct according to the twenty firstaspect of the invention comprising a nucleotide sequence having at least50% identity with SEQ ID NO: 88 operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an ELISA assay, Western blotting or, dependent onthe identity of the protein or polypeptide of interest, any suitableprotein identification and/or quantification assay known to the personskilled in the art. Preferably, the method of the invention allows foran increase in protein or polypeptide expression of at least 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%,1000%, 1500% or 2000% as compared to a method which only differs in thata construct is used in step a) that in said construct the nucleotidesequence having at least 50% identity with SEQ ID NO:88 has beenreplaced by an alternative sequence, preferably one of those asdescribed in example 11, more preferably when tested in a system asexemplified in example 11 which is enclosed herein. More specifically,preferably the expression of nucleotide sequence of interest encodingfor secreted alkaline phosphatase (SeAP) is measured in a mammalian cellsystem, most preferably in CHO cells, using a pcDNA3.1 expression vectorcomprising a nucleotide sequence having at least 50% identity with SEQID NO:88 operably linked to said nucleotide sequence of interest.Expression is preferably measured by measuring the conversion of anysuitable alkaline phosphatase substrate and expression levels arecompared to expression levels of said nucleotide sequence of interestwhich are measured under the same conditions except that in theexpression vector the nucleotide sequence having at least 50% identitywith SEQ ID NO:88 has been replaced by an alternative sequence,preferably one of those as described in example 11.

Twenty Fourth Aspect

In a twenty fourth aspect, the present invention provides a method forexpressing a protein or polypeptide of interest in an organism,comprising the steps of:

-   -   a) providing a nucleic acid construct according to the twentieth        aspect comprising a nucleotide sequence encoding a protein or        polypeptide of interest of the invention; and,    -   b) contacting a target cell and/or target tissue of an organism,        with said nucleic acid construct to obtain a transformed target        cell and/or transformed target tissue, allowing said transformed        cell to express the protein or polypeptide of interest; and        optionally,    -   c) allowing said transformed target cell to develop into a        transformed organism; and, optionally,    -   d) allowing said transformed organism to express the protein or        polypeptide of interest, for example, subjecting said        transformed organism to conditions leading to expression of the        protein or polypeptide of interest, and optionally recovering        said protein or polypeptide.

The target cell may be an embryonal target cell, e.g., embryonic stemcell, for example, derived from a non-human mammalian, such as bovine,porcine, et cetera species. Preferably, said target cell is not a humanembryonic stem cell. In the case of a multicellular fungus, such targetcell may be a fungal cell that can be proliferated into saidmulticellular fungus. When a transformed plant tissue or plant cell(e.g., pieces of leaf, stem segments, roots, but also protoplasts orplant cells cultivated by suspension) is obtained with this methodaccording to the invention, whole plants can be regenerated from saidtransformed tissue or cell in a suitable medium, which optionally maycontain antibiotics or biocides known in the art for the selection oftransformed cells. This method of the invention may be applied innucleic acid based vaccination and/or gene therapy preferably in amammal, most preferably in a human. Encompassed within the presentinvention is a method of treatment comprising the method of the presentaspect, wherein the protein or polypeptide of interest is a therapeuticand/or immunogenic protein or polypeptide. The invention also relates toa construct of the twentieth aspect of the invention for treatment,wherein the protein or polypeptide of interest is a therapeutic and/orimmunogenic protein or polypeptide. Furthermore, the invention relatesto the use of a construct of the twentieth aspect of the invention forthe manufacture of a medicament, wherein the protein or polypeptide ofinterest is a therapeutic and/or immunogenic protein or polypeptide.

Furthermore, an embodiment of the invention is a non-human transformedorganism. Said organism is transformed with a nucleotide sequence,recombinant nucleic acid construct, or vector according to the presentinvention, and is capable of producing the polypeptide of interest. Thisincludes a non-human transgenic organism, such as a transgenic non-humanmammalian, transgenic plant (including propagation, harvest and tissuematerial of said transgenic plant, including, but not limited to, leafs,roots, shoots and flowers), multicellular fungus, and the like.

Preferably, the method of this aspect of the invention allows for anincrease in expression of a protein or polypeptide of interest in saidorganism or at least in one tissue or organelle or organ of saidorganism. Preferably, expression levels are established in an expressionsystem using an expression construct according to the twenty firstaspect of the invention comprising a nucleotide sequence having at least50% identity with SEQ ID NO:88 operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest. Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an ELISA assay, Western blotting or, dependent onthe identity of the protein or polypeptide of interest, any suitableprotein identification and/or quantification assay known to the personskilled in the art. Preferably, this method of the invention allows foran increase in protein or polypeptide expression of at least 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%,1000%, 1500% or 2000% in said organism or at least in one tissue ororganelle or organ of said organism. as compared to a method which onlydiffers in that a construct is used in step a) wherein the nucleotidesequence having at least 50% identity with SEQ ID NO:88 has beenreplaced by an alternative sequence, preferably one of those asdescribed in example 11, preferably when tested in a system asexemplified in example 11 which is enclosed herein. More specifically,preferably the expression of a nucleotide sequence of interest encodingfor secreted alkaline phosphatase (SeAP) is measured in a mammalian cellsystem, most preferably in CHO cells, using a pcDNA3.1 expression vectorcomprising a nucleotide sequence having at least 50% identity with SEQID NO:88 operably linked to said nucleotide sequence of interest.Expression is preferably measured by measuring the conversion of anysuitable alkaline phosphatase substrate and expression levels arecompared to expression levels of said nucleotide sequence of interestwhich are measured under the same conditions except that in theexpression vector said nucleotide sequence having at least 50% identitywith SEQ ID NO:88 has been replaced by an alternative sequence,preferably one of those as described in example 11.

Twenty Fifth Aspect

In a twenty fifth aspect, the present invention provides a method fortranscription and optionally purifying the produced transcriptcomprising the step of:

-   -   a) providing a nucleic acid construct according to the twentieth        aspect comprising a nucleotide sequence of interest of the        invention; and,    -   b) contacting a cell with said nucleic acid construct to obtain        a transformed cell; and,    -   c) allowing said transformed cell to produce a transcript of the        nucleotide sequence of interest; and optionally,    -   d) purifying said produced transcript.

In a preferred embodiment of this method according to the invention anucleic acid construct as defined above in the twentieth aspect is used.The method of the invention may be an in vitro or ex vivo method. Themethod of the invention may be applied on a cell culture, organismculture, or tissue culture. The method of the invention may be appliedin nucleic acid based vaccination and/or gene therapy preferably in amammal, preferably in a human. Encompassed within the present inventionis a method for treatment comprising or consisting of the method of thepresent aspect, wherein the nucleotide sequence of interest encodes fora therapeutic transcript. The invention also relates to a construct ofthe twentieth aspect of the invention for use in treatment, wherein thenucleotide sequence of interest encodes for a therapeutic transcript.Furthermore, the invention relates to the use of a construct of thetwentieth aspect of the invention for the manufacture of a medicament,wherein the nucleotide sequence of interest encodes for a therapeutictranscript.

The skilled person is capable of transforming cells in accordance withstep b). Transformation methods as used in step b) include, but are notlimited to transfer of purified DNA via cationic lipid reagents andpolyethyleneimide (PEI), calcium-phosphate co-precipitation,microparticle bombardment, electroporation of protoplasts andmicroinjection or use of silicon fibers to facilitate penetration andtransfer of DNA into the host cell.

In step c) the transformed cell is allowed to produce a transcript ofthe nucleotide sequence of interest, and optionally the producedtranscript is subsequently recovered. For example, the transformed cellmay be subjected to conditions leading to transcription the nucleotidesequence of interest. The person skilled in the art is well aware oftechniques to be used for transcription the nucleotide sequence ofinterest. Methods in which the transformed cell does not need to besubjected to specific conditions leading to transcription of thenucleotide sequence of interest, but in which the nucleotide sequence ofinterest is automatically (e.g., constitutively) transcribed, are alsoincluded in the method of the present invention.

Purification steps depend on the transcript produced. The term“isolation” indicates that the transcript is found in a condition otherthan its native environment. In a preferred form, the isolatedtranscript is substantially free of other cellular components,particularly other homologous cellular components such as homologousproteins. It is preferred to provide the transcript in a greater than40% pure form, more preferably greater than 60% pure form. Even morepreferably it is preferred to provide the transcript in a highlypurified form, i.e., greater than 80% pure, more preferably greater than95% pure, and even more preferably greater than 99% pure, as determinedby Northern blot.

Preferably, the method of this aspect of the invention allows for anincrease in transcription of a nucleotide sequence of interest.Preferably, transcription levels are established in an expression systemusing an expression construct according to the second aspect of theinvention comprising a nucleotide sequence having at least 50% identitywith SEQ ID NO:88 operably linked to a nucleotide sequence of interest.Preferably, transcription of said nucleotide sequence of interest isdetected by a suitable assay such as RT-qPCR. Preferably, the method ofthe invention allows for an increase in transcription of at least 5%,10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,500%, 1000%, 1500% or 2000% as compared to a method which only differsin that a construct is used in step a) wherein said nucleotide sequencehaving at least 50% identity with SEQ ID NO:88 has been replaced by analternative sequence, preferably one of those as described in example11, preferably when tested in a system as exemplified in example 11which is enclosed herein. More specifically, preferably thetranscription of a nucleotide sequence of interest encoding for secretedalkaline phosphatase (SeAP) is measured in a mammalian cell system, mostpreferably in CHO cells, using a pcDNA3.1 expression vector comprising anucleotide sequence having at least 50% identity with SEQ ID NO:88operably linked to said nucleotide sequence of interest. Transcriptionis preferably measured using RT-qPCR and transcription levels arecompared to transcription levels of said nucleotide sequence of interestmeasured under the same conditions except that in the expression vectorused the nucleotide sequence having at least 50% identity with SEQ IDNO:88 has been replaced by an alternative sequence, preferably one ofthose as described in example 11.

Twenty Sixth Aspect

In an twenty sixth aspect, the present invention provides a use of anucleic acid molecule according to the nineteenth aspect of theinvention, and/or a use of a nucleic acid construct according to thetwentieth aspect of the invention, and/or a use of an expression vectoraccording to the twenty first aspect of the invention, and/or a use of acell according to the twenty second aspect of the invention, for thetranscription of a nucleotide sequence of interest and/or the expressionof a protein or polypeptide of interest.

Twenty Seven Aspect

In a twenty seven aspect, the present invention provides for a nucleicacid molecule according to according to the nineteenth aspect of theinvention, and/or a nucleic acid construct according to the twentiethaspect of the invention, and/or an expression vector according to thetwenty first aspect of the invention, and/or a cell according to thetwenty second aspect of the invention for use as a medicament. Theinvention also relates to a method of treatment comprising theadministration of a nucleic acid molecule according to the nineteenthaspect of the invention, and/or a nucleic acid construct according tothe twentieth aspect of the invention, and/or an expression vectoraccording to the twenty first aspect of the invention, and/or a cellaccording to the twenty second aspect of the invention, whereinpreferably said administration is to a mammal, more preferably to ahuman. Preferably, said treatment is nucleic acid based vaccinationand/or gene therapy preferably in a mammal, most preferably in a human.Furthermore, the invention relates to the use of a nucleic acid moleculeaccording to according to the nineteenth aspect of the invention, and/orthe use of a nucleic acid construct according to the twentieth aspect ofthe invention, and/or the use of an expression vector according to thetwenty first aspect of the invention, and/or the use of a cell accordingto the twenty second aspect of the invention, for the preparation of amedicament. Preferably said medicament is for nucleic acid basedvaccination and/or gene therapy preferably in a mammal, most preferablyin a human.

Definitions

The phrase “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. A nucleic acid of the invention is preferablymodified as compared to its naturally occurring counterpart bycomprising at least 1, 2, 3, 4, 5, 10, 20, 30 or 50 nucleotide mutationsas compared to its naturally occurring counterpart. Preferably, anucleic acid of the invention does not occur in nature. Nucleic acids ofthe invention can also include nucleotide analogs (e.g., BrdU), andnonphosphodiester internucleoside linkages (e.g., peptide nucleic acid(PNA) or thiodiester linkages). In particular, nucleic acids caninclude, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, ssRNA,dsRNA, non coding RNAs, hnRNA, premRNA, matured mRNA or any combinationthereof. The terms “nucleic acid sequence” and “nucleotide sequence” asused herein are interchangeable, and have their usual meaning in theart. The term refers to a DNA or RNA molecule in single or doublestranded form. An “isolated nucleic acid sequence” refers to a nucleicacid sequence which is no longer in the natural environment from whichit was isolated. A nucleic acid molecule is represented by a nucleotidesequence. Furthermore, an element such as, but not limited to anexpression enhancing element and a transcription regulating element, isrepresented by a nucleotide sequence.

A “recombinant construct” (or chimeric construct) refers to any nucleicacid sequence or molecule, which is not normally found in nature in aspecies, in particular a nucleic acid sequence, molecule or gene inwhich one or more parts of the nucleic acid sequence are present thatare not associated with each other in nature. For example, a recombinantconstruct comprises a promoter that is not associated in nature withpart or all of the transcribed region or with another regulating regioncomprised within said recombinant construct. The term “recombinantconstruct” is understood to include expression constructs in which apromoter or expression regulating sequence is operably linked to one ormore sense sequences (e.g. coding sequences) or to an antisense (reversecomplement of the sense strand) or inverted repeat sequence (sense andantisense, whereby the RNA transcript forms double stranded RNA upontranscription), or to any other sequence coding for a functional RNAmolecule.

A “nucleic acid construct” is defined as a polynucleotide which isisolated from a naturally occurring gene or which has been modified tocontain segments of polynucleotides which are combined or juxtaposed ina manner which would not otherwise exist in nature. Optionally, apolynucleotide present in a nucleic acid construct is operably linked toone or more control sequences, which direct the production ortranscription of a nucleotide sequence of interest and/or the expressionof a peptide or polypeptide of interest in a cell or in a subject

A “vector” or “plasmid” is herein understood to mean a man-made (usuallycircular) nucleic acid molecule resulting from the use of recombinantDNA technology and which is used to deliver exogenous DNA into a hostcell. Vectors usually comprise further genetic elements to facilitatetheir use in molecular cloning, such as e.g. selectable markers,multiple cloning sites and the like (see below). A nucleic acidconstruct may also be part of a recombinant viral vector for expressionof a protein in a plant or plant cell (e.g. a vector derived fromcauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or in amammalian organism or mammalian cell system (e.g. a vector derived fromMoloney murine leukemia virus (MMLV; a Retrovirus) a Lentivirus, anAdeno-associated virus (AAV) or an adenovirus (AdV)).

A “transformed cell” are terms referring to a new individual cell (ororganism), arising as a result of the introduction into said cell of atleast one nucleic acid molecule, especially comprising a chimeric orrecombinant construct encoding a desired protein or a nucleic acidsequence which upon transcription yields an antisense RNA for silencingof a target gene/gene family. The host cell may be a plant cell, abacterial cell (e.g. an Agrobacterium strain), a fungal cell (includinga yeast cell), an animal (including insect, mammalian) cell, etc. Thetransformed cell may contain the nucleic acid construct as anextra-chromosomally (episomal) replicating molecule, as anon-replicating molecule or comprises the recombinant constructintegrated in the nuclear or organellar DNA of the host cell. The term“organism” as used herein, encompasses all organisms consisting of morethan one cell, i.e. multicellular organisms, and includes multicellularfungi. “Transformation” and “transformed” refers to the transfer of anucleic acid sequence, generally a nucleic acid sequence comprising arecombinant construct or gene of interest (GOI), into the nuclear genomeof a cell to create a “transgenic” cell or organism comprising atransgene. The introduced nucleic acid sequence is generally, but notalways, integrated in the host genome. When the introduced nucleic acidsequence is not integrated in the host genome, one may speak of“transfection”, “transiently transfected”, and “transfected”. For thepurposes of the present patent specification, the terms“transformation”, “transiently transfected”, and “transfection” are usedinterchangeably, and refer to stable or transient presence of a nucleicacid sequence into a cell or organism. When the cell is a bacterialcell, the term usually refers to an extrachromosomal, self-replicatingvector which harbors a selectable antibiotic resistance.

“Sequence identity” or “identity” in the context of amino acid- ornucleic acid-sequence is herein defined as a relationship between two ormore amino acid (peptide, polypeptide, or protein) sequences or two ormore nucleic acid (nucleotide, polynucleotide) sequences, as determinedby comparing the sequences. In the art, “identity” also means the degreeof sequence relatedness between amino acid or nucleotide sequences, asthe case may be, as determined by the match between strings of suchsequences. Within the present invention, sequence identity with aparticular sequence indicated with a particular SEQ ID NO preferablymeans sequence identity over the entire length of said particularpolypeptide or polynucleotide sequence indicated with said particularSEQ ID NO. However, sequence identity with a particular sequenceindicated with a particular SEQ ID NO may also mean that sequenceidentity is assessed over a part of said SEQ ID NO. A part may mean atleast 50%, 60%, 70%, 80%, 90% or 95% of the length of said SEQ ID NO.The sequence information as provided herein should not be so narrowlyconstrued as to require inclusion of erroneously identified bases. Theskilled person is capable of identifying such erroneously identifiedbases and knows how to correct for such errors.

Any nucleotide sequences capable of hybridising to the nucleotidesequences of the invention are defined as being part of the cis-actingelements of the invention. Stringent hybridisation conditions are hereindefined as conditions that allow a nucleic acid sequence of at least 25,preferably 50, 75 or 100, and most preferably 150 or more nucleotides,to hybridise at a temperature of about 65° C. or of 65° C. in a solutioncomprising about 1 M salt or 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at 65° C. in asolution comprising about 0.1 M salt, or 0.1 M salt or less, preferably0.2×SSC or any other solution having a comparable ionic strength.Preferably, the hybridisation is performed overnight, i.e. at least for10 hours and preferably washing is performed for at least one hour withat least two changes of the washing solution. These conditions willusually allow the specific hybridisation of sequences having about 90%or more sequence identity or at least 90% sequence identity.

Moderate hybridization conditions are herein defined as conditions thatallow a nucleic acid sequence of at least 50, preferably 150 or morenucleotides, to hybridise at a temperature of about 45° C. or of 45° C.in a solution comprising about 1 M salt or 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength, and washing atroom temperature in a solution comprising about 1 M salt, or 1 M saltpreferably 6×SSC or any other solution having a comparable ionicstrength. Preferably, the hybridisation is performed overnight, i.e. atleast for 10 hours, and preferably washing is performed for at least onehour with at least two changes of the washing solution. These conditionswill usually allow the specific hybridisation of sequences having up to50% sequence identity. The person skilled in the art will be able tomodify these hybridisation conditions in order to specifically identifysequences varying in identity between 50% and 90%.

“Identity” can be readily calculated by known methods, including but notlimited to those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heine, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the GCG program package (Devereux,J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP,BLASTN, and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410(1990). The BLAST X program is publicly available from NCBI and othersources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). Thewell-known Smith Waterman algorithm may also be used to determineidentity.

Preferred parameters for polypeptide sequence comparison include thefollowing: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453(1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc.Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and GapLength Penalty: 4. A program useful with these parameters is publiclyavailable as the “Ogap” program from Genetics Computer Group, located inMadison, Wis. The aforementioned parameters are the default parametersfor amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following:Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970);Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap LengthPenalty: 3. Available as the Gap program from Genetics Computer Group,located in Madison, Wis. Given above are the default parameters fornucleic acid comparisons.

Preferred program and parameter for assessing identity for nucleic acidcomparison is calculated using EMBOSS Needle Nucleotide Alignmentalgorithm with the following parameters: DNAfull matrix with thefollowing gap penalties: open=10; extend=0.5 as carried out in example9.

The term “derived from” in the context of being derived from aparticular naturally occurring gene or sequence is defined herein asbeing chemically synthesized according to a naturally occurring gene orsequence and/or isolated and/or purified from a naturally occurring geneor sequence. Techniques for chemical synthesis, isolation and/orpurification of nucleic acid molecules are well known in the art. Ingeneral, a derived sequence is a partial sequence of the naturallyoccurring gene or sequence or a fraction of the naturally occurring geneor sequence. Optionally, the derived sequence comprises nucleic acidsubstitutions or mutations, preferably resulting in a sequence being atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical over its whole length to thenaturally occurring gene partial gene or sequence or partial sequence.

“Polypeptide” as used herein refers to any peptide, oligopeptide,polypeptide, gene product, expression product, or protein. A polypeptideis comprised of consecutive amino acids. The term “polypeptide”encompasses naturally occurring or synthetic molecules. A polypeptide isrepresented by an amino acid sequence. A polynucleotide is representedby a nucleotide sequence. A polypeptide is represented by an amino acidsequence.

The term “homologous” when used to indicate the relation between a givennucleic acid or polypeptide molecule and a given host organism or hostcell, is understood to mean that in nature the nucleic acid orpolypeptide molecule is produced by a host cell or organisms of the samespecies, preferably of the same variety or strain. If homologous to ahost cell, a nucleic acid sequence of interest, preferably encoding apolypeptide will typically be operably linked to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment.

When used to indicate the relatedness of two nucleic acid sequences theterm “homologous” means that one single-stranded nucleic acid sequencemay hybridise to a complementary single-stranded nucleic acid sequence.The degree of hybridisation may depend on a number of factors includingthe extent of identity between the sequences and the hybridisationconditions such as temperature and salt concentration as discussedlater. Preferably, the region of identity is greater than 5 bp, morepreferably the region of identity is greater than 10 bp.

The term “heterologous” when used to indicate the relation between agiven (recombinant) nucleic acid or polypeptide molecule and a givenhost organism or host cell, is understood to mean a nucleic acid orpolypeptide molecule from a foreign cell which does not occur naturallyas part of the organism, cell, genome or DNA or RNA sequence in which itis present, or which is found in a cell or location or locations in thegenome or DNA or RNA sequence that differ from that in which it is foundin nature. Heterologous nucleic acids or proteins are not endogenous tothe cell into which they are introduced, but have been obtained fromanother cell or synthetically or recombinantly produced.

When used to indicate the relatedness of two nucleic acid sequences, theterm the term “heterologous sequence” or “heterologous nucleic acid” isone that is not naturally found operably linked as neighboring sequenceof the other sequence. As used herein, the term “heterologous” may mean“recombinant”. “Recombinant” refers to a genetic entity distinct fromthat generally found in nature. As applied to a nucleotide sequence ornucleic acid molecule, this means that said nucleotide sequence ornucleic acid molecule is the product of various combinations of cloning,restriction and/or ligation steps, and other procedures that result inthe production of a construct that is distinct from a sequence ormolecule found in nature.

“Operably linked” is defined herein as a configuration in which acontrol sequence or regulating sequence is appropriately placed at aposition relative to the nucleotide sequence of interest, preferablycoding for the polypeptide of interest such that the control orregulating sequence directs or affects the transcription and/orproduction or expression of the nucleotide sequence of interest,preferably encoding a peptide or polypeptide of the invention in a celland/or in a subject. For instance, a promoter is operably linked to acoding sequence if the promoter is able to initiate or regulate thetranscription or expression of a coding sequence, in which case thecoding sequence should be understood as being “under the control of” thepromoter. When one or more nucleotide sequences and/or elementscomprised within a construct are defined herein to be “configured to beoperably linked to an optional nucleotide sequence of interest”, saidnucleotide sequences and/or elements are understood to be configuredwithin said construct in such a way that these nucleotide sequencesand/or elements are all operably linked to said nucleotide sequence ofinterest once said nucleotide sequence of interest is present in saidconstruct.

“Promoter” refers to a nucleic acid sequence located upstream or 5′ to atranslational start codon of an open reading frame (or protein-codingregion) of a gene and that is involved in recognition and binding of RNApolymerase II and other proteins (trans-acting transcription factors) toinitiate transcription. The term promoter refers to a nucleic acidfragment that functions to control the transcription of one or moregenes, located upstream with respect to the direction of transcriptionof the transcription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequences,including, but not limited to transcription factor binding sites,repressor and activator protein binding sites, and any other sequencesof nucleotides known to one skilled in the art to act directly orindirectly to regulate the amount of transcription from the promoter.The promoter does not include the transcription start site (TSS) butrather ends at nucleotide −1 of the transcription site, and does notinclude nucleotide sequences that become untranslated regions in thetranscribed mRNA such as the 5′-UTR. Promoters of the invention may betissue-specific, tissue-preferred, cell-type specific, inducible andconstitutive promoters. Tissue-specific promoters are promoters whichinitiate transcription only in certain tissues and refer to a sequenceof DNA that provides recognition signals for RNA polymerase and/or otherfactors required for transcription to begin, and/or for controllingexpression of the coding sequence precisely within certain tissues orwithin certain cells of that tissue. Expression in a tissue-specificmanner may be only in individual tissues or in combinations of tissues.Tissue-preferred promoters are promoters that preferentially initiatetranscription in certain tissues. Cell-type-specific promoters arepromoters that primarily drive expression in certain cell types.Inducible promoters are promoters that are capable of activatingtranscription of one or more DNA sequences or genes in response to aninducer. The DNA sequences or genes will not be transcribed when theinducer is absent. Activation of an inducible promoter is established byapplication of the inducer. Constitutive promoters are promoters thatare active under many environmental conditions and in many differenttissue types. Preferably, capability to initiate transcription isestablished in an expression system using an expression constructcomprising said promoter operably linked to a nucleotide sequence ofinterest using a suitable assay such a RT-qPCR or Northern blotting. Apromoter is said to be capable to start transcription if a transcriptcan be detected or if an increase in a transcript level is found of atleast 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,300%, 500%, 1000%, 1500% or 2000% as compared to transcription using aconstruct which only differs in that it is free of said promoter. In afurther preferred embodiment, capability to initiate expression isestablished in an expression system using an expression constructcomprising said promoter operably linked to a nucleotide sequenceencoding a protein or polypeptide of interest. Preferably, said proteinor polypeptide of interest is a secreted protein or polypeptide andexpression of said protein or polypeptide of interest is detected by asuitable assay such as an ELISA assay, Western blotting or, dependent onthe identity of the protein or polypeptide of interest, any suitableprotein identification and/or quantification assay known to the personskilled in the art. A promoter is said to be capable to initiateexpression if the protein or polypeptide of interest can be detected orif an increase in a expression level is found of at least 5%, 10%, 15%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 500%, 1000%,1500% or 2000% as compared to expression using a construct which onlydiffers in that it is free of said promoter. As a first and secondpromoter of the invention, an induced or constitutive promoter or acombination thereof may be used in the present invention.

An “intron” is a nucleotide sequence within a primary RNA transcriptthat is removed by RNA splicing or intron splicing while the finalmature RNA product is being generated. Assessment whether intronsplicing occurs can be done using any suitable method known to theperson skilled in the art, such as but not limited toreverse-transcriptase polymerase chain reaction (RT-PCR) followed bysize or sequence analysis of the RT-PCR product. Preferably, anucleotide sequence is an intron if at least 2%, 5%, 10%, 15%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90% or 100% of the primary RNA loses thissequence by RNA splicing using an assay suitable to detect intronsplicing as indicated above. Preferably, an intron comprises a splicesite GT at the 5′ end of the nucleotide sequence, and a splice site AGat the 3′ end of the nucleotide sequence, which splice site AG ispreceded by a pyrimidine rich nucleotide sequence or polypyrimidinetract, optionally separated from splice site AG by 1-50 nucleotides. Anintron may further comprise a branch site comprising the sequenceY-T-N-A-Y, at the 5′ side of the polypyrimidine tract. The branch sitemay have the nucleotide sequence C-Y-G-A-C. An “intronic sequence” isunderstood to be at least part of the nucleotide sequence of an intron.

“Expression” will be understood to include any step involved in theproduction of the peptide or polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification and secretion.

Optionally, a promoter represented by a nucleotide sequence present in anucleic acid construct is operably linked to another nucleotide sequenceencoding a peptide or polypeptide as identified herein.

An expression vector may be any vector which can be convenientlysubjected to recombinant DNA procedures and can bring about theexpression of a nucleotide sequence encoding a polypeptide of theinvention in a cell and/or in a subject.

As used herein, the “5′-UTR” is the sequence starting with nucleotide 1of the mRNA and ending with nucleotide −1 of the start codon. It ispossible that a regulating part of the promoter is comprised within thenucleotide sequence becoming a 5′-UTR; however, in such case, the 5′-UTRis still not part of the promoter as herein defined.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide or a polypeptide. Each control sequence may be native orforeign to the nucleic acid sequence harboring or encoding thepolynucleotide or the polypeptide. Such control sequences include, butare not limited to, a leader, optimal translation initiation sequences(as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), apolyadenylation sequence, a pro-peptide sequence, a pre-pro-peptidesequence, a promoter, a signal sequence, and a transcription terminator.At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals.

The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence, which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence, which shows transcriptionalactivity in the cell including mutant, truncated, and hybrid promoters,and may be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the cell.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence of interest, preferably encoding apolypeptide of interest. Any terminator, which is functional in thecell, may be used in the present invention.

The control sequence may also be a suitable leader sequence, anon-translated region of a mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence of interest, preferably encoding apolypeptide of interest. Any leader sequence, which is functional in thecell, may be used in the present invention.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add adenine residues to transcribed mRNA. Any polyadenylationsequence, which is functional in the cell, may be used in the presentinvention.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a product or a composition or anucleic acid molecule or a peptide or polypeptide of a nucleic acidconstruct or vector or cell as defined herein may comprise additionalcomponent(s) than the ones specifically identified; said additionalcomponent(s) not altering the unique characteristic of the invention. Inaddition, reference to an element by the indefinite article “a” or “an”does not exclude the possibility that more than one of the elements ispresent, unless the context clearly requires that there be one and onlyone of the elements. The indefinite article “a” or “an” thus usuallymeans “at least one”.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

TABLE 1 Sequence identification SEQ ID NO: Description  1 Expressionenhancing element 1  2 Expression enhancing element 2  3 UN1    4-13sequences derived from UN1 14 UN2 15 UN1dGAA 16 UN2dGAA 17 R3 18 fUN1 19UN2-2 20 UN2-3 21 UN2-4 22 UN2-5 23 UN2-6 24 UN2-7 25 UN2-8 26 UN2-9 27UN2-10 28 UN1dGAA-2 29 UN1dGAA-3 30 UN1dGAA-4 31 UN1dGAA-5 32 UN1dGAA-633 UN2dGAA-2 34 UN2dGAA-3 35 UN2dGAA-4 36 UN1shuffle 37 UN1shuffle-2 38UN1shuffle-3 39 UN1shuffle-4 40 UN1shuffle-5 41 UN1shuffle-6 42UN2shuffle-1 43 UN2shuffle-2 44 CAA1 45 CAA2 46 CAA3 47 CAA4 48 CAA5 49CAA6 50 TATA1 51 TATA2 52 CMV promoter enhancer sequence 53 UBC enhancerregion 54 CMV promoter enhancer sequence 55 construct 56 construct 57CMV promoter sequence 58 Minimal CMV promoter sequence 59 EEE1-Xt 60EEE1-80 61 EEE1-60 62 EEE1-50 63 EEE1-SL 64 HC RACE primer 65 Lightchain vector sequence 66 Heavy chain vector sequence 67 HuMabl proteinlight chain 68 HuMabl protein heavy 69 HuMab2 protein light chain 70HuMab2 protein and heavy chain 71 pcDNA3.1 (+) 72 SeAP protein 73 EEE1 +CMV + TEE 74 EEE1-Xt + CMV + TEE 75 EEE1-80 + CMV + TEE 76 EEE1-60 +CMV + TEE 77 pPNic384 78 pPNic602 insert 79 EF1a promoter 80 EEE1-A1 81EEE1-A2 82 EEE1-A3 83 EEE1-B1 84 EEE1-B2 85 EEE1-B3 86 EEE1-B4 87EEE1-B5 88 Transcription regulating sequence

FIGURES

FIG. 1. Schematic map of intronic promoter construct and differenttranscripts. The construct comprises 2 promoters. Transcription byPromoter 1 results in a primary transcript including the intron thatcontains the complete Promoter 2 sequence and is bordered by 5′ and3′-splice sites. After intron splicing, said primary transcript resultsin a mRNA without said intron (Transcript 1) encoding a “Gene”.Transcription from Promoter 2 also results in a mRNA (Transcript 2)encoding the same “Gene”.

FIGS. 2a -2 b. Schematic map of EEE1 (FIG. 2a ) and EEE2 (FIG. 2b )elements showing some features of the UBC and CCT8 genes relevant totheir promoter activity in a genomic context. Features include thepredicted transcription start site (TSS), 5′-UTRs, exon and introninformation.

FIG. 3. Schematic map of an expression vector for an Ig light chain(IgLC) with the EEE1 sequence integrated upstream of the CMV promoter.

FIGS. 4a -4 b. Comparison of HuMab1 production by CHO-S pools stablytransfected with Reference or EEE1 constructs. Expression vector without(FIG. 4a ) and with (FIG. 4b ) additional expression regulating element.The bars represent the average exhaust titers of 4 pools derived from 2independent transfections. Pools were grown in 125 ml shake-flasks in 30ml CD FortiCHO selection medium.

FIG. 5. Comparison of HuMab1 production by CHO-S pools stablytransfected with Reference or EEE2 constructs. The bars represent theaverage exhaust titers of 4 pools derived from 2 independenttransfections. Pools were grown in 125 ml shake-flasks in 30 ml CDFortiCHO selection medium.

FIG. 6. Analysis of HuMab1 production by top-10 CHO-S clonal cell linesstably transfected with EEE1-TEE constructs harboring EEE1. Cells weregrown in 125 ml shake-flasks in 30 ml CD FortiCHO selection medium.

FIGS. 7a -7 b. Comparison of HuMab2 production by CHO-S pools stablytransfected with EEE1 in reference vector (FIG. 7a , left panel) and invector with additional regulating element (FIG. 7a , right panel). Thebars represent the average exhaust titers of 4 pools derived from 2independent transfections. Pools were grown in 125 ml shake-flasks in 30ml CD FortiCHO selection medium. (FIG. 7b ) Analysis of HuMab2production by top-12 Reference and EEE1-TEE CHO-S clonal cell lines.Cells were grown in 125 ml shake-flasks in 30 ml CD FortiCHO selectionmedium.

FIG. 8. Comparison of SeAP activity in the exhaust media of CHO-S poolsstably transfected with Reference or EEE1 constructs. Expression vectorwithout (left panel) and with (right panel) additional expressionregulating element. The bars represent the average activities of 4 poolsderived from 2 independent transfections, measured using the SEAPReporter Gene Assay Kit, Abcam. Pools were grown in 125 ml shake-flasksin 30 ml CD FortiCHO selection medium.

FIG. 9. Comparison of SeAP activity in the exhaust media of CHO-S poolsstably transfected with constructs containing different versions of theEEE1 element. The bars represent the average activities of 4 poolsderived from 2 independent transfections, measured using the SEAPReporter Gene Assay Kit, Abcam. Pools were grown in 125 ml shake-flasksin 30 ml CD FortiCHO selection medium.

FIGS. 10a -10 b. 5 ′-RACE amplification of 5′-ends of heavy chaintranscripts from CHO-S clones stably transfected with EEE1-TEEconstructs expressing HuMab1 (FIG. 10a ). Two bands are detected onagarose gel, corresponding to the transcripts generated by the CMVpromoter (Transcript 1) and the UBC promoter (Transcript 2). The sizedifference between Transcript 1 and Transcript 2 is explained in aschematic map of intronic promoter construct and different transcripts(FIG. 10b ). The construct comprises 2 promoters, UBC and CMV. The CMVpromoter is linked to a TEE sequence which also comprises a shortintron. Transcription by the CMV promoter results, after intronsplicing, in mRNAs with TEE as 5′-UTR (Transcript 1). The UBC promoteris linked to a partial UBC 5′-UTR region which comprises a 5′ splicedonor site and precedes the CMV promoter. Transcription by the UBCpromoter results in mRNAs with the UBC 5′-UTR sequence (Transcript 2).The large intron which is spliced from the primary transcript runs fromthe 5′-splice donor sequence in the UBC sequence to the 3′-spliceacceptor site in the TEE and contains the complete CMV sequence.

FIG. 11. Effect of EEE in Pichia pastoris expressing recombinant humaninterleukin 8. Each bar represents the average expression of 10independent clones.

The invention will be explained in more detail in the following Examplessection, with reference to the appended figures. The examples serve forillustration purposes only, and do not intend to limit the presentinvention in any way.

EXAMPLES

The expression enhancing element represented by SEQ ID NO: 1 is based onthe Chinese hamster (Cricetulus griseus) ubiquitin-C (UBC) gene. Itcomprises the predicted promoter sequence and part of the5′-untranslated region (FIG. 2A). The expression enhancing elementrepresented by SEQ ID NO: 2 is based on the human CCT8 gene (chaperonincontaining TCP1, subunit 8). It comprises the predicted promotersequence and part of the 5′-untranslated region as well as a shortsequence encoding 27 amino acids and part of the first intron (FIG. 2B).

Example 1

Expression plasmids were constructed based on the pcDNA3.1 expressionvector (SEQ ID NO: 71). The vector was modified by removing the f1-ori.Coding sequences for an IgG1 (HuMab1) heavy (represented by a sequencethat is at least 96% identical SEQ ID NO: 68) and light chain(represented by a sequence that is at least 99% identical SEQ ID NO: 67)genes were inserted in this vector (the light chain coding sequence wasinserted in the vector represented by SEQ ID NO: 65 and the heavy chaincoding sequence was inserted in the vector represented by SEQ ID NO:66), resulting in the Reference constructs. To generate the EEE1(Expression Enhancing Element 1) vectors, the EEE1-sequence (SEQ IDNO: 1) was inserted upstream of the CMV promoter (SEQ ID NO: 57) (FIG.3). EEE2 (SEQ ID NO: 2) was introduced in a similar way, resulting inthe EEE2 vectors. A vector with an additional expression regulatingelement was generated by replacing the pcDNA3.1 5′-UTR for SEQ ID NO: 19(transcription enhancing element; TEE).

CHO-S cells (Life Technologies) were maintained per manufacturer'sinstructions. Duplicate transfections were performed using 3E7 cells, 50μg of linearized DNA and FreeStyle MAX Reagent (Life Technologies).Post-transfection pools were split in two and selected in CD FortiCHOmedium supplemented with 8 mM glutamine and 800 μg/ml G418. Selectedpools were seeded in 30 ml of the same medium at a density of 3E5cells/ml in 125 ml shake-flasks. The HuMab1 exhaust titers weredetermined by ELISA (FIG. 4). Exhaust titers of the Reference pools weretoo low for accurate determination. This indicates that the antibody ispoorly expressed. The EEE1 pools produced approximately 1 μg/ml (FIG.4A). A similar effect was observed in the vector with an additionalexpression regulating element. In this vector the introduction of EEE1increased the production from approximately 0.2 μg/ml to 9-12 μg/ml instable pools from three independent transfection experiments (FIG. 4B).These data show that a poorly expressed antibody can be expressed atsignificantly higher levels by introduction of the EEE1 element.

Example 2

The effect of introducing the EEE2 element was studied in a vectorharboring an additional expression regulating element (See Example 1,FIG. 4B). CHO-S cells were transfected either with the reference or withthe EEE2 constructs as described previously. Antibody exhaust titers ofthe stably transfected EEE2 pools were over 20 times higher than theReference pools (FIG. 5).

Example 3

The EEE1-TEE and Reference pools generated previously were seeded in six96-well plates at a density of 0.5 cell/well in CD FortiCHO selectionmedium. The Reference cells showed impaired growth as compared to theEEE1-TEE clones and thus no HuMab1 was produced. Clones of EEE1-TEEshowed normal growth and HuMab1 production (See below). 100 ClonalEEE1-TEE lines were assessed for HuMab1 production in microtiter plates.The 10 clones with highest specific productivity were expanded to 125 mlshake-flasks. The clones were seeded in 30 ml of CD FortiCHO selectionmedium at a density of 3E5 cells/ml and HuMab1 exhaust titers weredetermined by ELISA (FIG. 6). Clones produced up to 0,25 μg/ml HuMab1.These data indicate that the EEE1 can facilitate the generation ofclonal lines and allows the generation of clonal lines with relevantexpression levels.

Example 4

The copy number of antibody expressing EEE comprising clones wasdetermined. The PrimerExpress program (Life Technologies) was used todesign Taqman primers and probes specific for the heavy- and lightchains of HuMab1 and β-2 microglobulin. The primers were combined in atriplex Taqman assay to measure gene copies in gDNA samples of EEE1-TEEHuMab1 clones and pools. The gene copy numbers were compared with HuMab1titers (Table 2). Clonal cell lines producing similar HuMab1 titers haddifferent numbers of light and heavy chain gene copies (Clone 1 and 2).Also, clones producing very different HuMab1 titers had similar genecopy numbers (Clone 3 and 4). In pools relatively high numbers of lightand heavy chain genes were paired with relatively low expression levels.These data (Table 2) indicate that there is no correlation between EEEcomprising gene copy number and HuMab1 expression levels.

TABLE 2 Titers of IgG1 and gene copy numbers IgG titer LC HC Clone1123.7 36.8 25.1 Clone2 118.2 1.7 1.7 Clone3 143.6 3.1 1.2 Clone4 7.2 4.60.7 Pool 9.0 17.5 21.1

Example 5

The HuMab1 heavy and light chain genes of the previous examples werereplaced by heavy and light chain genes (SEQ ID NO's: 69 and 70)encoding a biosimilar antibody (HuMab2 derived from DrugBank AccessionNumber DB00072). The constructs were used to generate CHO-S pools asdescribed previously. Using ELISA, the exhaust titers were determined.The data (6.3 μg/ml without enhancing element) indicate that thisantibody is produced to a higher level than the antibody from theprevious examples. Without any additional expression regulating elementintroduction of the EEE resulted in a 3.7 fold increase (FIG. 7A, leftpanel), in the modified vector the increase is 7 fold (FIG. 7A, rightpanel). Since stand-alone the additional expression regulating elementresults in a 40% increase, the data also indicate a synergistic effectbetween the EEE and the additional expression regulating element. Clonallines were isolated from the Reference and EEE1-TEE pools as describedpreviously. The best EEE1-TEE clones produced 3-fold higher HuMab2titers as compared to the best Reference clones (FIG. 7B). These dataindicate that the EEE1 element can be successfully applied in enhancingrecombinant protein expression from stable cell lines.

Example 6

The HuMab1 light chain gene of the constructs from Example 1 wasreplaced by the gene encoding secreted alkaline phosphatase (SeAP; SEQID NO: 72). The constructs were used to generate CHO-S pools asdescribed previously. The SeAP activity was measured in the exhaustmedium using the SEAP Reporter Gene Assay Kit, Abcam. The EEE1 poolsshowed 2-fold higher activity as compared to the Reference pools (FIG.8). In the EEE1-TEE pools the increase was almost 4-fold as compared tothe Reference pool. These data show that EEE1 enhances the expression ofa single subunit non-antibody protein in a transfected cell line.

Example 7

The SeAP constructs used in Example 6 all comprised the CMV promoter.Two TEE vector variants were made that contained the human EF-1αpromoter instead of CMV (SEQ ID NO: 79). The constructs were used togenerate CHO-S pools as described previously. The SeAP activity wasmeasured in the exhaust medium using the SEAP Reporter Gene Assay Kit,Abcam. The EEE1-TEE pool with EF-1α as intronic promoter produced2.8-fold higher SeAP activity as compared to the Reference EF-1αpromoter pool without EEE1. These data show that EEE1 enhances theexpression of a protein in an intronic promoter construct when theintronic promoter is not the CMV promoter, such as the EF-1α promoter.

Example 8

The EEE1 element of the EEE1 SeAP-expression vector was replaced by thefollowing variants: 1. EEE1-80 represented by SEQ ID NO: 60 has a 290 bptruncation from the 5′-end; 2. EEE1-60 represented by SEQ ID NO: 61 witha 580 bp truncation from the 5′-end; 3. EEE1-50 represented by SEQ IDNO: 62 with a 725 bp truncation from the 5′-end; 4. EEE1-Xt representedby SEQ ID NO: 59 with a 800 bp extension from the genomic C. griseus UBCsequence at the 5′-end; 5. EEE1-SL (SEQ ID NO: 63) has all majorpredicted splice donor and acceptor sites mutated. SeAP activity in thesupernatant of cells transfected with the EEE1 element was set at 100%,which decreased to 39% activity without the EEE1 element (FIG. 9). The5′ truncations of the EEE1-80 and EEE1-60 constructs gradually decreasedactivity but still showed enhanced activity as compared to the No-EEEconstruct. The EEE1-50 element decreased SeAP activity to 40%, which issimilar to the No-EEE construct. The EEE1-Xt construct showed almost 40%increased activity as compared to the EEE1 construct. The data suggestthat sequences with more than 50% identity to EEE1 can function asexpression enhancing elements. The EEE1-Xt construct produced almost 40%increased activity as compared to the EEE1 construct, which shows thatadditional enhancer sequences reside in the region upstream of thegenomic sequence from which EEE1 was taken. The activity of the EEE1element is severely impaired by 4 nt mutations of the EEE1-SL constructwhich prevent correct intron splicing, resulting in a significantreduction in SeAP expression as compared to the EEE1 construct.

Example 9

The EEE1 element of the EEE1 SeAP-expression vector was replaced by 9variants of the EEE1 element, which can be grouped based on 2 differenttypes of mutations. The first type of EEE1 variants (EEE1-A) all hadchanges within the EEE1 or EEE1-Xt element with more than 30 percent ofnucleotides mutated, each in another of the 3 regions which eachconsisted of at least 244 bp. The second type of EEE1 variants (EEE1-B)also had the same size as the EEE1 element (1,449 bp) with at least 96percent sequence identity, with mutations that targeted differentfunctional sequences of the EEE1 sequence. The different mutations arelisted in Table 3.

TABLE 3 Modifications of EEE1 Type A: More than 30% mutated in 3 regionsof EEE1 Identity to Modified Size modified Variant SEQ ID NO: EEE1¹⁾EEE1 region region (bp) EEE1-A1 80 71.6%²⁾ 5′ promoter region 1,526EEE1-A2 81 95.0%  3′ promoter region 244 EEE1-A3 82 81.8%  intron region480 Type B: Mutations that target specific domains of EEE1 Identity toModification of Variant SEQ ID NO: EEE1¹⁾ EEE1 sequence EEE1-B1 83 97.9%1: 7 nt changed in nt 144-152 2: 4 nt changed in nt 612-615 3: 4 ntchanged in nt 667-670 4: 6 nt changed in nt 816-823   5: 5 nt changed innt 1,106-1,112   6: 5 nt changed in nt 1,432-1,438 EEE1-B2 84 96.5% 50single bp mutations = 50% of CG's mutated in nt 227-1,409; predictedtranscription factor binding sites maintained EEE1-B3 85 99.7% 5 singlebp mutations = 50% of CG's mutated in nt 549-603 EEE1-B4 86 96.5% 50single bp mutations = 50% of CG's mutated in nt 227-1,409; 8 predictedsites for transcription factors SP1, HSF, and NFκB affected. EEE1-B5 8796.0% 51 bp mutations in 12 regions with predicted transcription factorbinding activity spanning nt 105-1,449 were mutated.  EEE1-B6³⁾ 63 99.7%4 single bp mutations eliminating predicted and known splice-donor oracceptor sites, including known donor site (nt 970), nt 545 and 552 inpromoter region, nt 1,267 in intron region. ¹⁾Identity calculated usingEMBOSS Needle Nucleotide Alignment algorithm with the followingparameters: DNAfull matrix with the following gap penalties: open = 10;extend = 0.5 ²⁾% identity EEE1-A1 calculated relative to EEE1-Xt ³⁾Thisis referred to as EEE1-SL in Example 8

SeAP activity in the supernatant of cells transfected with the differentvariants was measured. Activity of cells with EEE1 element was used asreference (100%). Without EEE1 element the activity was 24% in thisexperiment. SeAP activity of cells transfected with the EEE1-A2 andEEE1-A3 constructs was decreased to 75% and 48% relative to EEE1,respectively (Table 4). This is higher than the 24% activity observedwith the No-EEE construct in this experiment. The EEE1-A1 constructdecreased SeAP activity to 30% relative to the EEE1-Xt construct onwhich it is based, which is still higher than the No-EEE construct whichproduces only 18% of SeAP activity relative to the EEE1-Xt construct.The data show that EEE1 variants with as little as 72% overall identityand locally 50% identity to the genomic UBC sequence can function asexpression enhancing elements.

SeAP activity of cells transfected with the EEE1-B1 to B6 constructs wasdecreased by up to 42% relative to the EEE1 construct (Table 4). Thedata show that mutations in regions with a predicted functionality inthe intronic promoter activity of the EEE1 element can significantlylimit the expression enhancement capability of the EEE1 element. Forinstance, mutating 4 nt involved in intron-splicing resulted in 38%decreased SeAP titers (EEE1-B6). Mutation of different sets of CpG'salso resulted in decreased SeAP titers (B1, B2, B4).

TABLE 4 SeAP activity of EEE1 variants Construct Activity relative toEEE1 (%)¹⁾ No-EEE1 24 EEE1 100 EEE1-A2 75 EEE1-A3 48 EEE1-B1 58 EEE1-B260 EEE1-B3 73 EEE1-B4 58 EEE1-B5 84 EEE1-B6 62 Construct Activityrelative to EEE1-Xt (%)¹⁾ No-EEE1 18 EEE1-Xt 100 EEE1-Al 30 ¹⁾Valuesrepresent the average activities of 4 pools derived from 2 independenttransfections, measured using the SEAP Reporter Gene Assay Kit, Abcam.Pools were grown in 125 ml shake-flasks in 30 ml CD FortiCHO selectionmedium

Example 10

A EEE1-TEE CHO-S clone from Example 3 was grown and cells were harvestedin log-phase. Total RNA was isolated from the cells using AllPrepDNA/RNA Mini Kit (Qiagen). cDNA was synthesized using the EpicentreExact Start Eukaryotic mRNA 5′ and 3′ RACE Kit. First strand cDNA wasamplified using the 5′ RACE primer from the kit combined with a heavychain specific primer (SEQ ID NO: 64) and ZymoTaq DNA polymerase. ThePCR product was analyzed on 1.2% agarose gels, showing two discretebands (FIG. 10A) which were separately isolated and inserted in aPCR4-TOPO vector (Life Technologies). Sequencing analysis revealed thatthe upper band seen on the agarose gel corresponds to the transcriptinitiated from the CMV promoter. The lower band corresponds to thetranscript initiated from the UBC promoter. Both products have thepredicted intronic sequence spliced out correctly. The differences insize correspond to the different lengths of the 5′-UTRs, as depicted inFIG. 10B. The data show that both promoters contribute to transcription.

Example 11

CHO-S pools stable transfected with constructs with three differentsingle promoters were compared by the SeAP activity in the supernatant.Pools were grown in 125 ml shake-flasks in 30 ml CD FortiCHO selectionmedium and SeAP activity was measured using the SEAP Reporter Gene AssayKit (Abcam) in 4 pools per construct derived from 2 independenttransfections. The constructs either contained the CMV promoter (Example6), the EF-1α promoter (Example 7), or the UBC promoter (Example 11).The UBC promoter produced 2.7-fold higher SeAP activity as compared tothe CMV promoter construct. The UBC promoter produced 6.0-fold higherSeAP activity as compared to the EF-1α promoter. The data shows that theexpression with the UBC promoter alone is higher as compared to the CMVpromoter or the EF-1α promoter alone.

Example 12 Methanol Induced Secretion of IL8 in Pichia pastoris GS115Integrative Transformants

Plasmids for stable transformation of Pichia pastoris with humaninterleukin 8 (hIL-8) expression constructs were generated in plasmidpPIC9K (Life Technologies). Insertion of the hIL-8 gene in pPIC9Kresulted in plasmid pPNic384 (SEQ ID NO: 77), which contains the hIL-8gene under control of the AOX1 promoter. The EEE1 sequence was insertedupstream of the AOX1 promoter as a AatII-AleI fragment (SEQ ID NO: 78)in pPNic384, resulting in plasmid pPNic602.

The expression vectors were linearized by digestion with SalI andtransformed into P. pastoris strain GS115 using electroporation asrecommended (Invitrogen, 2008). Transformants were plated on RDB agarplates (Regeneration Dextrose Medium, a medium lacking histidine). Afterincubation at 30° C. for 48 h, large colonies were observed. A controltransformation without DNA was performed resulting in no colonies.Randomly 10 clones per construct were picked from the transformationplate and grown to saturation in 800 μl BMG (Buffered minimal mediumwith 1% glycerol), in 2 ml deep well plates. The plate was kept in ashaking incubator (Infors-HT Microton) set at 30° C., 1000 rpm for 18hours. The optical density of the culture was between 5-10 absorbanceunits at 600 nm. The cells were harvested and the medium replaced by 800μl of BMM (Buffered minimal medium with 0.5% methanol) in 2 ml deep wellplates. The cells were grown in the shaking incubator and every 24 hours0.5% methanol (final concentration) was added to the culture to maintaininduction. After 72 hours of methanol induction the culture supernatantwere collected and assayed for secreted hIL8 yields using the AlphaLISAhIL8 kit (Perkin Elmer). The data show (FIG. 11) that there is asignificant difference between the IL8 yields of the reference and theEEE1 transformants, suggesting that the EEE1 sequence upstream of thepromoter improves the hIL8 yields compared to expression plasmid withoutthe EEE1 sequence.

1.-15. (canceled)
 16. A nucleic acid construct comprising a firstpromoter, a second promoter, and a single nucleotide sequence ofinterest, wherein said first promoter and second promoter areconstitutive promoters and are both operably linked to said singlenucleotide sequence of interest, and wherein said second promoter is anintronic promoter flanked by a first intronic sequence located upstreamof said second promoter and a second intronic sequence locateddownstream of said second promoter, and wherein said single nucleotidesequence of interest is under the control of said first promoter andsaid second promoter.
 17. The nucleic acid construct according to claim1, wherein said nucleic acid construct further comprises an additionalexpression regulating sequence, and wherein said additional expressionregulating sequence, said first promoter and said second promoter areall operably linked to said nucleic acid sequence of interest.
 18. Thenucleic acid construct according to claim 1, wherein said nucleotidesequence of interest encodes a protein or polypeptide of interest. 19.An expression vector comprising the nucleic acid construct according toclaim
 1. 20. An in vitro cell comprising the nucleic acid constructaccording to claim
 1. 21. A non-human cell comprising the nucleic acidconstruct according to claim
 1. 22. The nucleic acid construct accordingto claim 2, wherein said additional expression regulating sequencecomprises or consists of an intron.
 23. The nucleic acid constructaccording to claim 3, wherein said protein or polypeptide of interest isa heterologous protein or polypeptide.
 24. The nucleic acid constructaccording to claim 1, wherein said first promoter comprises a sequencehaving at least 50% identity to SEQ ID NO: 1 over its whole length orhaving at least 50% identity to SEQ ID NO: 2 over its whole length. 25.The nucleic acid construct according to claim 1, wherein said secondpromoter comprises a sequence having at least 95% identity to SEQ ID NO:57, 58, or 79 over its whole length.
 26. The nucleic acid constructaccording to claim 1, wherein said second promoter comprises a sequencehaving at least 95% identity to SEQ ID NO: 57 or 58 over its wholelength.
 27. The nucleic acid construct according to claim 1, whereinsaid first promoter comprises a sequence having at least 50% identity toSEQ ID NO: 1 or 2 over its whole length and said second promotercomprises a sequence having at least 95% identity to SEQ ID NO: 57, 58or 79 over its whole length.
 28. The nucleic acid construct according toclaim 1, wherein said first promoter comprises a sequence having atleast 50% identity to SEQ ID NO: 1 over its whole length and said secondpromoter comprises a sequence having at least 95% identity to SEQ ID NO:57 or 58 over its whole length.
 29. The nucleic acid construct accordingto claim 1, wherein said first promoter comprises a sequence having atleast 50% identity to SEQ ID NO: 2 over its whole length and said secondpromoter comprises a sequence having at least 95% identity to SEQ ID NO:57 or 58 over its whole length.
 30. The nucleic acid construct accordingto claim 1, wherein said second promoter is a human or murinecytomegalovirus (CMV) promoter.
 31. The nucleic acid construct accordingto claim 1, wherein said first promoter and said first intronic sequencecomprise the sequence of SEQ ID NO:
 1. 32. The nucleic acid constructaccording to claim 1, wherein said second intronic sequence comprisesthe sequence of SEQ ID NO:19.
 33. A method for transcription andoptionally purifying the produced transcript comprising the steps of: a)providing a nucleic acid construct comprising a first promoter, a secondpromoter, and a single nucleotide sequence of interest, wherein saidfirst promoter and second promoter are constitutive promoters and areboth operably linked to said single nucleotide sequence of interest, andwherein said second promoter is an intronic promoter flanked by a firstintronic sequence located upstream of said second promoter and a secondintronic sequence located downstream of said second promoter, andwherein said single nucleotide sequence of interest is under the controlof said first promoter and said second promoter, b) contacting a cellwith said nucleic acid construct to obtain a transformed cell; and, c)allowing said transformed cell to produce a transcript of the nucleotidesequence of interest; and optionally, d) purifying said producedtranscript.
 34. A method for transcription and optionally purifying theproduced transcript comprising the steps of: a) providing a nucleic acidconstruct comprising in the 5′ to 3′ direction an expression enhancingelement, a heterologous promoter and a nucleotide sequence of interest,wherein said expression enhancing element has at least 50% identity toSEQ ID NO: 1 or 2 over its whole length, and wherein said expressionenhancing element and said heterologous promoter are operably linked toa same, single nucleotide sequence of interest; and, b) contacting acell with said nucleic acid construct to obtain a transformed cell; and,c) allowing said transformed cell to produce a transcript of thenucleotide sequence of interest; and optionally, d) purifying saidproduced transcript.